Vaccine

ABSTRACT

The invention relates to a vaccine for the treatment of disease caused by  Neisseria , the vaccine including one or more immunogenic components for  Neisseria  serogroups, as well as antibodies to the immunogenic components and methods of preventing and treating  Neisseria  infections. The immunogens are based on elements of the inner core lipopolysaccharide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application entitled“Vaccine”, filed on Jul. 11, 2002 and assigned Ser. No. 10/089,583,which claimed the benefit of PCT/GB00/03758 filed Oct. 2, 2000, whichclaimed the benefit of U.S. provisional patent application 60/196,305,filed Apr. 12, 2000 and U.S. provisional patent application 60/156,940,filed Sep. 30, 1999.

The present invention relates to vaccines against Neisseria infection,especially to infection by pathogenic Neisseria meningitidis andNeisseria gonorrhoeae.

BACKGROUND OF THE INVENTION

Septicaemia and meningitis caused by Neisseria meningitidis remain aglobal health problem, especially in young children. Neisseriameningitidis is usually a commensal of the nasopharynx, the only majornatural reservoir of this organism. The virulence factors thatpotentiate the capacity of Neisseria meningitidis to cause invasivedisease include capsular polysaccharides, pili (fimbrae) or outermembrane proteins and lipopolysaccharides (DeVoe, I. W. 1982. MicrobiolRev 46: 162-190, Jennings, H. J. 1989. Contrib Microbiol Immunol 10:151-165, Tonjum, T., and M. Koomey. 1997. Gene 192: 155-163, Nassif, X.,et al. 1997. Gene 192: 149-153, Poolman, J. T. 1996. Adv Exp Med Biol397: 73-33, Verheul, A. F., et al. 1993. Microbiol Rev 57: 34-49,Preston, A, et al. 1996 Crit. Rev Microbiol 22:139-180).

Existing licensed vaccines against capsular serogroups A, C, W and X areavailable (Frasch, C. E. 1989. Clin Microbiol Rev 2 Suppl: S134-138,Herbert, M. A., et al. 1995. Commun Dis Reg CDR Rev 5: R130-135,Rosenstein, N., et al. 1998. J.A.M.A. 279: 435-439), but generally lacksatisfactory immunogenicity in very young children and do not inducelong lasting protective immunity (Peltola, H., et al. 1977. N Engl J Med297: 686-691, Peltola, H., et al. 1985. Pediatrics 76: 91-96, Reingold,A. L., C. V. Broome, et al. 1985. Lancet II:114-118, Lepow, M. L., etal. 1986. J Infect Dis 154: 1033-1036, Cadoz, M. 1998. Vaccine 16:1391-1395). Nonetheless, their utility has been significant in affordingprotection to selected populations such as the military, travelers andthose at exceptional risk in outbreaks or epidemics (CDC. 1990. MMWRMorb Mortal Wkly Rep 39, No. 42: 763). Very recently, meningococcalconjugate Group C vaccines have been introduced as a routineimmunization in the United Kingdom.

The major public health priority concerning invasive meningococcalinfections is to identify Group B vaccines that are highly effective ininfants and give long term protection. Group B strains have accountedfor a substantial, often a majority of invasive Neisseria meningitidisinfections in many countries in Europe and North America (CDR. 1997April. Communicable Disease Weekly Report. 7, No. 14). Prevention ofGroup B invasive disease represents a particularly difficult challengein vaccine development as the capsular polysaccharide is very poorlyimmunogenic and even conjugates have shown disappointing immunogenicity(Jennings, H. J., and H. C. Lugowski. 1981. J. Immunology127:1011-1018). Further, there are concerns about the safety of vaccineswhose rationale is to induce antibodies to the Group B polysaccharide, ahomopolymer of α-linked 2-8 neuraminic acid. The identicalpolysialicacid (PSA) is a post translational modification of aglycoprotein present on human cells, especially neurons, the latter isreferred to as neural cell adhesion molecule (N-CAM) (Finne, J., et al.1983. Lancet 2: 355-357). Both theoretical and experimental evidencehave been used to argue that the induction of antibodies might result inauto-immune, pathological damage to host tissues.

Alternative approaches to develop vaccine candidates against Group BNeisseria meningitidis are being actively explored. These include: outermembrane porins (Poolman, J. T., et al. 1995. Meningococcal disease, p.21-34K. Cartwright (ed.). John Wiley and sons, Wetzler, L. M. 1994. AnnN Y Acad Sci 730: 367-370, Rosenqvist E., et al. 1995. Infect Immun63:4642-4652, Zollinger, W. D., et al. 1997. Infect Immun 65:1053-1060), transferrin binding proteins (Al'Aldeen, A. A., and K. A.Cartwright. 1996. J Infect 33: 153-157) and lipopolysaccharides(Verheul, A. F., et al. 1993. Infect Immun 61: 187-196, Jennings, H. J.,et al. 1984. Infect Immun 43: 407-412, Jennings, H. J., et al. 987.Antonie Van Leeuwenhoek 53: 519-522, Gu, X. X., and C. M. Tsai. 1993.Infect Immun 61: 1873-1880, Moxon, E. R., et al. 1998. Adv Exp Med Biol435: 237-243).

The structure of Neisseria meningitidis LPS has been studied inconsiderable detail by Jennings H. and co-workers with additionalcontributions by others (Griffiss, J. M. et al. 1987. Infect Immun 55:1792-1800, Stephens, D. S., et al. 1994. Infect Immun 62: 2947-2952,Apicella, M. A., et al. 1994. Methods Enzymol 235: 242-252, Poolman, J.T. 1990. Polysaccharides and membrane vaccines, p. 57-86. In Bacterialvaccines, A. Mizrahi (ed.)., et al. 1997. FEMS Microbiol Lett 146:247-253). The structures of major glycoforms for several immunotypes(L1-L9) have been published L1, L6 (Di Fabio, J. L., et al. 1990. Can JChem 68: 1029-1034, Wakarchuk, W. W., et al. 1998. Eur J Biochem 254:626-633); L3 (Pavliak, V., et al. 1993. J Biol Chem 268: 14146-14152);L5 (Michon, F., et al. 1990. J. Biol Chem 265:7243-7247); L2 (Gamian,A., et al. 1992. J Biol Chem 267: 922-925); L4, L7 (Kogan, G., et al.1997. Carbohydr Res 298: 191-199): L8 (Wakarchuk, W. W., et al., 1996,J. Biol. Chem. 271, 19166-19173), L9 (Jennings, H. J., et al. 1983.Carbohydr. Res. 21: 233-241). Reference is also made to the followingdiscussion of the accompanying FIG. 1.

It is known that, in addition to this inter-strain variation, individualNeisseria meningitidis strains exhibit extensive phase variation ofouter core LPS structures (reviewed in van Putten, J. P., and B. D.Robertson. 1995. Mol Microbiol 16: 847-853 and Andersen, S. R., et al.1997 Microb Pathog 23: 139-155). The molecular mechanism of this intrastrain variation involves hypermutable loci within the reading framesencoding several glycosyl transferases (Gotschlich, E. C. 1994. J ExptMed 180: 2181-2190, Jennings, M. P., et al. 1995. Mol Microbiol 18:729-740). Similar mechanisms of phenotypic variation have been reportedfor other phase-variable surface components of pathogenic Neisseria,including Opc (Sakari, J., et al. 1994. Mol. Microbiol 13: 207-217), Opa(Stem, A., et al. 1986. Cell 47: 61-71) and PilC proteins (Jonsson, A.B., et al. 1991. EMBO J. 10: 477-488). The high frequency, reversiblemolecular switching is mediated by homopolymeric tracts of cytosines orguanines through slippage-like mechanisms that results in frame shifts(Gotschlich, E. C. 1994. J Expt Med 180: 2181-2190, Jennings, M. P., etal. 1995. Mol Microbiol 18: 729-740, Stern, A., and T. F. Meyer. 1987.Mol. Microbiol 1: 5-12).

Despite the extensive antigenic variation of LPS, the inner core of theLPS has been considered to be relatively highly conserved, and thereforethe use of the inner core of the LPS structure has been suggested foruse in vaccine design. However, the problems with candidate vaccinegeneration in this way are numerous.

First, although it was known that certain components of the inner corecould be immunogenic (Jennings, H. J. Lugowski, C. and Ashton, F. E.1984. Infect. Immun. 43: 407-412 and Verheul, A. F., et al., 1991.Infect. Immun. 59: 3566-3573), the extent of conservation of theseepitopes across the diversity of meningococcal disease isolates was notknown and evidence of bactericidal activity of antibodies to theseepitopes has not been shown. U.S. Pat. No. 5,705,161 discloses thatoligosaccharides of meningococcal immunotypes differ, for example, withregard to monosaccharide composition, amount and location ofphosphoethanolamine groups and degree of acetylation of the inner coreGlcNAc unit or other units, indicating that many possible structures maybe found in the core structure. U.S. Pat. No. 5,705,161 also suggeststhat a portion of the core of a meningococcal LPS may be suitable foruse in a vaccine, although no specific immunogenic epitopes orsupporting data are disclosed.

Secondly, given the presence of the outer core LPS structure and othersurface exposed non-LPS structures, including capsule, it is not knownwhether the inner core structure is accessible to the immune system toallow a bactericidal immune response to be generated. Furthermore, anyvaccine would need to contain immunogenic structures which elicit animmune response to the complete range of pathogenic Neisseriameningitidis strains. However, the extent of variation exhibited by theinner core structure of virulent strains is not known, and rigorousinvestigation of the problem has not been undertaken.

Furthermore, in the publication New Generation Vaccines [1997, Ed. M. M.Levine, publ. Marcel Deker Inc, New York, Chapter 34, page 481], it isstated that, with respect to vaccine development, ‘including LPS thatconsists only of the common inner core region of the oligosaccharide maynot result in induction of bactericidal antibodies . . . ”.

In addition, other species of the genus Neisseria pose global healthproblems. For example, Neisseria gonorrhoeae is involved in sexuallytransmitted diseases such as urethritis, salpingitis, cervicitis,proctitis and pharyngitis, and is a major cause of pelvic inflammatorydisease in women.

Accordingly, there is still a need in the art for an effective vaccineagainst pathogenic Neisseria infection, such as Neisseria meningitidisand Neisseria gonorrhoeae infection.

The present invention sets out to address this need.

STATEMENT OF INVENTION

In a first aspect, the invention relates to a vaccine for the treatmentof disease caused by Neisseria infection, the vaccine comprising animmunogenic component of Neisseria strains. The vaccine presents aconserved and accessible epitope that in turn promotes a functional andprotective response.

We have now discovered that the inner core of the LPS of Neisseria canbe used to generate a protective immune response to Neisseriainfections, for example Neisseria meningitidis infections. Forsimplicity the present invention is herein exemplified principally bydiscussion of vaccines and treatments against Neisseria meningitidisinfections, but the invention extends to diseases caused by otherpathogenic Neisseria species.

Using a range of novel monoclonal antibodies, epitopes belonging to theinner core of Neisseria meningitidis have been identified which havebeen found to be accessible to the immune system, and which are capableof stimulating the production of functional, protective antibodies.Moreover, analysis of Neisseria meningitidis strains using the newantibody tools indicates that certain epitopes are common to a range ofNeisseria meningitidis disease isolates, and sometimes occur in amajority of such strains. Accordingly a vaccine comprising only alimited range of Neisseria meningitidis inner core epitopes can provideeffective immunoprophylaxis against the complete range of strainscausing Neisseria meningitidis infection. Similar considerations applyto other pathogenic species.

In a related aspect, the invention provides a vaccine effective againststrains of the bacteria of the genus Neisseria, especially strains ofthe species Neisseria meningitidis. Particularly in the latter instance,the vaccine comprises one or more immunogens which can generateantibodies that recognize epitopes in encapsulated strains. The one ormore immunogens represent one or more accessible inner core epitopes.Thus, the immunogens can give rise to antibodies that recognize amajority of strains.

We use the word “principal” to refer to a majority. Thus, a principalimmunogenic component elicits antibodies to a majority of strains.

In our approach, antibodies were generated by immunizing mice usingNeisseria meningitidis galE mutants. The antibodies produced werespecific to the LPS inner core because galE mutants lack outer corestructures. The reactivity of these antibodies against a panel ofNeisseria meningitidis strains representative of the diversity found innatural populations of disease isolates was investigated. One monoclonalantibody reacted with 70% of all Neisseria meningitidis strains tested,suggesting strong conservation of the inner core epitope recognized bythis antibody, termed antibody B5. The epitope against which B5 reactshas been characterized and can be used to form the basis of a vaccine toprevent Neisseria infections.

A hybridoma producing the monoclonal antibody B5, designated hybridomaNmL3B5, has been deposited under the Budapest Treaty on 26 Sep. 2000with the International Depositary Authority of Canada in Winnipeg,Canada, and given the Accession Number IDAC 260900-1.

In this way, we have obtained proof in principle that one or more of theinner core epitopes of LPS are conserved and accessible to antibodies,that a specific immune response to these epitopes can mediateprotection, and that LPS inner core oligosaccharides can be candidatevaccines. The inner core LPS typically consists of an inner coreoligosaccharide attached to lipid A, with the general formula as shown:

where R1 is a substituent at the 3-position of HepII, and is hydrogen orGlc-α-(1, or phosphoethanolamine; R2 is a substituent at the 6-positionof HepII, and is hydrogen or phosphoethanolamine; R.3 is a substituentat the 7-position of HepII, and is hydrogen or phosphoethanolamine, andR4 is acetyl or hydrogen at the 3-position, 4-position or 6-position ofthe GlcNAc residue, or any combination thereof; and where Glc isD-glucopyranose; Kdo is 3-deoxy-D-manno-2-octulosonic acid; Hep isL-glycero-D-manno-heptose, and GlcNAc is2-acetamido-2-deoxy-D-glucopyranose.

GENERAL DESCRIPTION OF THE INVENTION

The principal immunogenic component for Neisseria meningitidis strainsis preferably a single immunogenic component found in at least 50% ofNeisseria meningitidis strains, i.e. in the majority of naturallyoccurring Neisseria meningitidis strains. The principal immunogeniccomponent forms a candidate vaccine immunogen. Preferably theimmunogenic component of the vaccine of the present invention is any oneelement or structure of Neisseria meningitidis or other species ofNeisseria capable of provoking an immune response, either alone or incombination with another agent such as a carrier. Preferably theprincipal immunogenic component comprises of or consists of an epitopewhich is a part or all of the inner core structure of the Neisseriameningitidis LPS. The immunogenic component may also be derived fromthis inner core, be a synthetic version of the inner core, or be afunctional equivalent thereof such as a peptide mimic. The inner coreLPS structure of Neisseria meningitidis is generally defined as thatshown in FIG. 1 and as outlined in the figure legend. The immunogeniccomponent is suitably one which elicits an immune response in thepresence and in the absence of outer core LPS.

The principal immunogenic component is conserved in at least 50% ofNeisseria strains within the species, preferably at least 60%, and morepreferably at least 70%. Reactivity with 100% strains is an idealizedtarget, and so the immunogenic component typically recognizes at most95%, or 85% of the strains. Conservation is suitably assessedfunctionally, in terms of antibody cross-reactivity. We prefer that theimmunogenic component is present in at least 50% of serogroup B strains,preferably at least 60%, more preferably at least 70%, even morepreferably at least 76%. Suitably, assessment of the cross reactivity ofthe immunogenic component is made using a representative collection ofstrains, such those outlined in Maiden (Maiden M. C. J., et al., 1998,P.N.A.S. 195, 3140-3145).

Preferably the principal immunogenic component is found in the Neisseriameningitidis immunotype L3, and preferably it is not in L2. Morespecifically, we prefer that the immunogen is found in the immunotypesL1, L3, L7, L8 and L9, but not in L2, L4, L5 or L6. In other words, weprefer that the immunogen, notably the principal immunogenic component,generates antibodies which are reactive with at least the L3 immunotype,and usually the L1, L3, L7, L8 and L9 immunotypes, but not with L2, andusually not the L2, L4, L5 and L6 immunotypes. There are conformationaldifferences forced on the inner core of the L2 and L3 immunotypes bydifferent arrangements at HepII, namely the PEtn moiety at the6-position in L2 or at the 3-position in L3, and the Glc residue at the3-position in L2. Currently we do not envisage the possibility of asingle epitope for both L2 and L3 immunotypes. In other words, withoutdismissing the possibility of a single epitope, the present invention isexpected to require different immunogens to elicit antibodies for L2 andL3.

Preferably the principal immunogenic component is a conserved epitope onthe LPS inner core recognized by an antibody termed B5 herein. Thepreferred epitope of the invention isthmus any epitope recognized by theB5 antibody.

Preferably the immunogenic component is a conserved epitope on the LPSinner core defined by the presence of a phosphoethanolamine moiety(PEtn) linked to the 3-position of HepII, the β-chain heptose, of theinner core, or is a functional equivalent thereof. In this respect.Where the context permits, HepI and HepII refer to the heptose residuesof the inner core oligosaccharide which respectively are proximal anddistal to the lipid A moiety of the neisserial LPS, without beingnecessarily tied to the general formula given above.

Preferably this epitope comprises a glucose residue on HepI, the α-chainheptose residue. While this glucose is not necessary for B5 biding, itis required for optimal recognition.

The principal immunogenic component of the present invention ispreferably an epitope on the LPS inner core which comprises an N-acetylglucosamine on HepII. The presence of N-acetyl glucosamine is requiredfor optimal recognition by B5.

Preferably the principal immunogenic component comprises both theN-acetyl glucosamine on HepII and a glucose residue on HepI.

The immunogenic component of the present invention is typically onlylimited by the requirement for a phosphoethanolamine moiety (PEtn)linked to the 3-position of HepII of the inner core, which is requiredfor B5 reactivity. The structure of the inner core may be modified,replaced, or removed, as necessary, to the extent that these are notneeded. Similarly, any outer core structures may be modified or deleted,to the extent that structural elements are not needed. There is norequirement for the immunogenic component to lack the outer coreportion, or equivalent, of the LPS. The immunogenic component maycomprise outer core elements having a galactose component, for examplethe terminal galactose residue of the lacto-N-neotetraose. In onesuitably embodiment, the immunogenic component is derived from LPS andis free from other cellular material. Alternatively, cellular materialmay be present, and can take the form of live or killed bacteria.

In a related aspect, the vaccine of this invention has an immunogenicepitope recognized by an antibody to a galE mutant of Neisseriameningitidis.

In a further embodiment the vaccine suitably comprises furtherimmunogenic elements from the inner core with an aim to achieving up to100% coverage. Preferably the vaccine comprises only a limited number(4-6, or less) of immunogenic elements, more preferably only thoseglycoforms which are representative of all possible PEtn positions onHepII, the β-chain heptose, of the inner core, i.e. wherein PEtn is atthe 3-position, exocyclic (6-position or 7-position) or absent, with orwithout an α-1-3 linked glucose at HepII, or a combination thereof. Thepresence of PEtn substituent is not required for the generation ofantibodies by an immunogenic component of this invention.

Moreover, as detailed herein, the epitopes of this invention areimmunogenic and accessible, and thus can be used to develop an effectivevaccine. Furthermore, as detailed herein, a vaccine containing only alimited number of glycoforms (representing all the possible PEtnpositions on HepII, namely position 3-, or 6-, or 7- or none, andcombinations thereof), is able to effectively provide protection againstthe diverse range of meningococcal isolates causing invasive disease.

Accordingly the vaccine of the present invention preferably comprises anepitope which is defined by the presence of a phosphoethanolamine moiety(PEtn) linked to the 3-position of HepII of the inner core, andadditionally comprises an epitope defined by the presence of PEtn on the6-position of HepII of the inner core, and/or an epitope defined by PEtnon the 7-position of HepII of the inner core, or wherein there is noadditional PEtn addition. Preferably the vaccine contains onlyimmunogenic components which are these inner core glycoform variants.

The B5 antibody of the present invention also recognizes the inner corestructures of Neisseria gonorhoeae and Neisseria lactamica. As such, theinvention extends to any Neisseria species, and any reference toNeisseria meningitidis can as appropriate be extended to other Neisseriaspecies, preferably Neisseria meningitidis, Neisseria gonorrhoeae andNeisseria lactamica, most preferably Neisseria meningitidis. Theinvention also extends to immunogenic components in other Neisseriaspecies which are related to those identified in Neisseria meningitidis,either by function, antibody reactivity or structure. The invention isnot limited to pathogenic strains of Neisseria. The vaccine of thisinvention can be derived from a commensal strain of Neisseria,especially a strain of Neisseria lactamica. The species Neisserialactamica is typically strongly immunogenic, and therefore we preferthat the LPS inner core immunogenic component is derived from thisspecies.

The vaccine may thus be homologous or heterologous, and thus founded onan immunogenic component from the target micro-organism, homologous, orfrom a different micro-organism, heterologous. The micro-organism can benaturally occurring or not, such as can be produced by recombinanttechniques. In particular, the micro-organism can be engineered tomodify the epitope or to modify other components.

In a further aspect of the invention we have determined that a secondmonoclonal antibody, herein termed A4, is able to react with inner coreepitopes of nearly all of the Neisseria meningitidis strains which donot react with the B5 antibody. Thus, of the 100 Neisseria meningitidisstrains tested, 30% were not reactive with B5 and were found to lack aPEtn moiety at the 3-position of HepII. Of these 30 strains, 27 werereactive with A4. Accordingly, a vaccine comprising only 2 inner coreepitopes, corresponding to those epitopes defined by cross reactivitywith A4 and B5, provides 97% coverage of a representative collection ofNeisseria meningitidis strains, preferably as assessed by using thecollection of strains as outlined in Maiden et al. [supra]. A preferredepitope of the invention is thus also any epitope recognized by the A4antibody.

A hybridoma producing the monoclonal antibody A4, designated hybridomaNmL4galEA4, has been deposited under the Budapest Treaty on 26 Sep. 2000with the International Depositary Authority of Canada in Winnipeg,Canada, and given the Accession Number IDAC 260900-2.

The present invention thus also relates to a vaccine comprising a fewimmunogenic components, wherein at least 70% of Neisseria meningitidisstrains of the species possess at least one of the immunogeniccomponents, preferably 80%, preferably 90%, and most preferably 97%. Inthis way the vaccine can give protective coverage against Neisseriainfection in 70%, preferably 80%, 90% or even 97% or more of cases.

A few immunogenic components suitably means at least two immunogeniccomponents, preferably only 2. More generally the few componentscomprise 2 to 6 components, such as 2, 3, 4, 5 or 6 components, moresuitably 2, 3 or 4 components. Preferably the immunogenic components area few glycoforms of the inner core, representative of all naturalNeisseria meningitidis strains. In this way, a vaccine containing alimited number of glycoforms can give approaching 100% coverage ofNeisseria meningitidis strains.

A representation of the 3D structures of the LPS inner core having aPEtn moiety at the 3-position, 6-position or absent at HepII are shownin FIG. 3. Accordingly, the present invention also extends toimmunogenic elements which have the same or similar structures to theseinner core structures, as defined by their 3D geometry and to antibodiescapable of interacting with such structures, either as assessed invitro, in vivo or in silico.

The immunogenic elements of the invention are preferably those shown toelicit antibodies having opsonic and bactericidal activity, and shown togenerate antibodies which confer passive protection in in vivo models.

The invention also extends to use of any immunogenic element as definedabove in the preparation of a medicament for the prevention, treatmentor diagnosis of Neisseria infection.

The candidate vaccine immunogens of the present invention may be suitedfor the prevention of all Neisseria infections. However, a vaccine forthe treatment of Neisseria meningitidis is preferred, with a vaccine forgroup B strains especially preferred.

Preferably the immunogenic element of the vaccine is accessible in thepresence of bacterial capsule. Accordingly, antibodies generated by anindividual who is vaccinated will be able to access the same epitope oninvading strains of Neisseria, and thus protect the individual frominfection. Antibodies given directly to a patient for treatment, alsoare thus able to directly access the target Neisseria strains.

Preferably the vaccine of the present invention comprises epitopes whichare capable of stimulating antibodies which are opsonic. We furtherprefer that these antibodies are capable of binding to wild typeNeisseria strains to confer protection against infection and which arebactericidal.

The present invention also provides a method for treating pathogenicNeisseria. The method employs one or a few immunogenic components whichgive rise to effective antibodies. And which rely on an inner coreepitope for stimulating the immune response. The immune response isordinarily B cell mediated, but we can include T cell mediated immunity.The antibodies generated by the vaccine of this invention bind to innercore elements of the pathogenic target bacterium.

Diseases caused by Neisseria meningitidis include principallymeningitis, septicaemia and pneumonia, and the prevention and treatmentof these diseases is especially preferred in the present invention.Diseases caused by Neisseria gonorrhoeae include sexually transmitteddiseases such as urethritis, cervicitis, proctitis pharyngitis,salpingitis, epididymitis and bacteremia/arthritis. Additionally, theinvention extends to treatment and prevention of any other disease whichresults from Neisseria infection, especially to diseases in whichNeisseria infection could weaken the immune system such that anotherdisease or pathogen could be harmful to an individual. The treatment canbe preventative or curative.

The vaccine of the present invention is a formulation suitable for safedelivery to a subject, allowing the subject to develop an immuneresponse to future infection by Neisseria. Vaccines of the presentinvention are preferably formulated vaccines in which any of theimmunogenic components of the vaccine may be conjugated, and anysuitable agent for conjugation may be used. Conjugation enablesmodification of the presentation of the antigen, and may be achieved byconventional techniques. Examples of agents for conjugation includeproteins from homologous or heterologous species. In this way, theimmunogenic component of the present invention forms a saccharidepeptide conjugate. Preferably the peptide portion comprises a T cellactivating epitope.

The vaccines of the present invention may be delivered with an adjuvant,to enhance the immune response to the immunogenic components. Suitableadjuvants include aluminium salts, oils in combination with bacterialmacromolecules, liposomes, muramyl dipeptide, ISCOMS, bacterial toxinssuch as pertussis, cholera and those derived from E. coli and cytokinessuch as IL-1, IL-2 and IFNγ.

The vaccine of the invention may be delivered by suitable means, such asby oral delivery or parenteral administration, injection, nutraceuticalor other delivery means, and may be provided in any suitable deliveryform such as tablets, pills, capsules granules, solutions, suspensionsor emulsions. Suitably the vaccine components are prepared in the formof asterile, isotonic solution.

The present invention also extends to the monoclonal antibodies derivedfrom the concepts and methodologies described herein, including but notlimited to B5 and A4, and use of these antibodies in the treatment ofNeisseria infection. The invention also relates to pharmaceuticalpreparations comprising such antibodies in combination withpharmaceutically acceptable carrier. Such preparations may be deliveredby any suitable means, such as those exemplified above for vaccinedelivery, and used in combination with other active agents or adjuvants.

The correct dosage of the antibody or vaccine will vary according to theparticular formulation, mode of application, and the particular hostbeing treated. Factors such as age, body weight, sex, diet, and time ofadministration, rate of excretion, condition of the host, drugcombinations, and reaction sensitivities are suitably to be taken intoaccount.

The antibodies and vaccines compositions of the present invention may beused with other drugs to provide combination treatments. The other drugsmay form part of the same composition, or be provided as a separatecomposition for administration at the same time or a different time.

In addition to the antibodies themselves, the invention also relates tothe hybridomas which produce such antibodies.

Antibodies against the immunogenic components of the invention may begenerated by administering the immunogenic components to an animal,preferably a non-human animal using standard protocols. For thepreparation of monoclonal antibodies, any suitable techniques can beused. Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce appropriate single chainantibodies. Moreover, transgenic mice or other organisms or animal maybe used to express humanized antibodies immunospecific to theimmunogenic components of the invention.

Alternatively, other methods, for example phage display technology maybe used to select antibody genes for proteins with binding activitiestowards immunogenic components of the present invention.

Antibodies of the invention may be either monoclonal or polyclonalantibodies, as appropriate.

The present invention also relates to a method for the prevention ofNeisseria infection, the method comprising administering to a subject inneed of such treatment an effective amount of a vaccine as describedabove. Preferably the administration is adequate to produce a longlasting antibody and/or T cell immune response to protect the subjectfrom infection, particularly Neisseria meningitidis infection.

The invention also relates to a method for the treatment of Neisseriainfection, the method comprising administering to a subject in need ofsuch treatment an effective amount of an antibody to the Neisseriameningitidis inner core. Preferably, the antibody is B5 or A4, or anantibody which recognizes the same epitope as B5 or A4, or an antibodyderived from the concepts and methodologies herein described, or is acombination thereof.

Moreover, the methods of the invention may be extended to identificationof epitopes in any bacterial strain. Epitopes so identified may betested both for accessibility, conservation across the population andfunctional activity, using methods as outlined in the attached Examples.The present invention thus additionally relates to a method for theidentification of an immunogenic element, comprising raising an antibodyto a bacterial structure, preferably bacterial LPS structure, morepreferably a bacterial inner core LPS structure, and testing the epitoperecognized by the antibody far accessibility to antibody in the wildtype strain optionally also comprising testing the epitope forconservation across the bacterial population and testing for functionalactivity to the epitope in vivo.

Preferably the bacterial species are Neisseria species, preferablyNeisseria meningitidis, Neisseria gonorrhoeae or Neisseria lactamica.

Specifically, the present invention provides a method to generateantibodies to the inner core of Neisseria meningitidis. For the firsttime it has been possible to screen a population of Neisseriameningitidis strains to identify whole population features which areindependent of immunotype.

Accordingly, the present invention also relates to a method for theidentification of immunogenic epitopes of Neisseria meningitidis, themethod comprising the steps of:

1 generating antibodies to the inner core of Neisseria meningitidis, byinoculation of host organism with a galE mutant strain of Neisseriameningitidis, and2 testing such antibodies against a wild type Neisseria meningitidisstrain to identify those antibodies which are reactive, and for whichthe epitopes are therefore accessible.

The potential utility of epitopes so identified may be further assessedby screening antibodies which react with the inner core of Neisseriameningitidis galE strain against a panel of strains which arerepresentative of strain diversity. Preferably the strain panel isselected using an approach based upon a population analysis. Epitopes soidentified may then be tested in functional assays, as outlined inExample 3.

In particular the invention extends to a method for the analysis ofantibody biding to bacteria wherein natural isolates of bacteria arestudied when grown on and adherent to tissue cultured cells, such asHUVECs. This assay provides a monolayer of cells to which the bacteriaadhere in a biologically relevant environment. Previous attempts usingNeisseria, for example, directly adherent to gelatin- or matrigel-coatedcoverslips resulted in low numbers of adherent bacteria after repeatedwashings and high non-specific background staining. In particular weprefer that the antibody biding is analyzed using confocal microscopy.

This method also identifies antibodies suitable for therapeutic use, andthe invention extends to such antibodies.

Moreover, key biosynthetic genes for each step in LPS synthesis havebeen identified (Preston et al., 1996, Crit. Rev. Microbiol. 22,139-180) and this allows the construction of a series of mutants fromwhich LPS glycoforms of varying size and complexities can be madeavailable to facilitate the identification of conserved epitopes (vander Ley et al., 1997, FEMS Microbiol. Letter 146, 247-253, Jennings etal 1993, Mol. Microbiol. 361-369, Jennings et al., 1995, Microb Pathog19, 391-407, van der Ley et al., 1996, Mol Microbiol 19, 1117-1125).

The present invention also relates to the gene found in Neisseriameningitidis which is involved in PEtn substitution at the 3-position onHepII, and to genes related in structure and function. As yet no geneshave been identified in any bacteria that are involved in addition ofPEtn to LPS structures. Using B5, specific for an inner core LPS epitopecontaining a PEtn, we have identified a putative LPS phosphoethanolaminetransferase gene (designated hypo3) in Neisseria meningitidis. Hypo3 wasnamed arbitrarily by us, as it is the 3rd reading frame in a fragment ofDNA selected by experimentation, from the MC58 genome sequence. Theoriginal hypo3 is therefore from MC58. This ORF is called NMB2010 in theTIGR data base (MC58 genome sequence) and although designated as aprotein of unknown function, they classify it as a “YhbX/YhjW/YijP/YjdBfamily protein”. This indicates that homologues have been inferred inother organisms but they do not know the function of them. The homologuein the serogroup A sequence at the Sanger Centre is designated NMA0431,although this gene is smaller than hypo3. Hypo3 is involved in PEtnsubstitution at the 3-position at HepII. Furthermore, the presence ofthe complete gene is required for the expression of the B5 reactivephenotype in Neisseria meningitidis as well as other pathogenic andcommensal Neisseria species.

The identification of the gene allows mutants to be created which areisogenic apart from hypo3, and which differ only in the presence orabsence of PEtn at the 3-position of HepII in the LPS inner core. Suchstrains can be used in comparative studies. Moreover, strainsappropriate for vaccine production can be engineered so that theycomprise the preferred PEtn structure at the 3-position, or engineeredso that this PEtn cannot be present.

Accordingly, the invention relates to use of the hypo3 gene, orhomologue thereof, in the production of a Neisseria strain for theassessment, treatment or prevention of Neisseria infection. Thehomologue may have 60%, 70%, 80%, 90% or more homology or identity tohypo3, as assessed at the DNA level. Use of the gene includes themethods outlined above, for preparing genetically modified strains forvaccination, isolation of appropriate epitopes and generation of strainsfor comparative studies. More generally, we envisage the identificationand use of any gene which plays a role in the biosynthetic pathway, andwhich has an effect on the conservation, accessibility or function ofthe immunogen.

The present invention is now illustrated by the following Figures andExamples which are not limiting upon the present invention, wherein:

FIG. 1 illustrates the LPS structure of various Neisseria meningitidisimmunotypes;

FIG. 2 illustrates cross reactivity of B5 with selected immunotypes andmutants of Neisseria meningitidis LPS;

FIG. 3 illustrates molecular models of the calculated (MMC) lowestenergy states of the core oligosaccharide from galE mutants of L3, L4and L8 dephosphorylated;

FIG. 4 illustrates cross reactivity of B5 with genetically modified L3LPS and chemically modified L8 LPS from Neisseria meningitidis;

FIG. 5 illustrates confocal immunofluorescence microscopy of Neisseriameningitidis organisms strain MC58 adherent to HUVECs;

FIG. 6 illustrates silver stained tricine gels of LPS preparations fromNeisseria meningitidis group B strains not reactive with B5;

FIG. 7 illustrates accessibility of the LPS epitope to A4 in Neisseriameningitidis whole cells;

FIG. 8 illustrates conservation of the LPS epitope across Neisseriameningitidis serogroups;

FIG. 9 illustrates the strategy for the Example 2;

FIG. 10 illustrates ELISA titres of antibodies to L3 galE LPS (IgG) inpaired sera taken early and late from children with invasivemeningococcal disease, and mean % phagocytosis of Neisseria meningitidisMC58 with paired sera taken early and late from children with invasivemeningococcal disease with human peripheral blood mononuclear cells andhuman complement;

FIG. 11 a illustrates mean % phagocytosis of Neisseria meningitidis MC58with MAb B5 pre-incubated with increasing concentrations of either (i)B5 reactive or (ii) B5 non-reactive galE LPS with human peripheral bloodpolymorphonuclear cells and human complement;

FIG. 11 b illustrates mean % phagocytosis of pair of Neisseriameningitidis wild-type isogenic strains (Neisseria meningitidis BZ157)that are either MAb B5 reactive or B5 non-reactive with MAb B5 as theopsonin with human peripheral blood mononuclear cells and humancomplement;

FIG. 11 c illustrates mean % phagocytosis of fluorescent latex beadscoated with either purified LPS from L3 galE mutant (10 μg/ml) oruncoated, in the presence of MAb B5 or final buffer, with humanperipheral blood mononuclear cells and human complement;

FIG. 12 illustrates mean % survival of Neisseria meningitidis galEmutant in the presence and absence of MAb B5 against two-fold serialdilutions of human pooled serum starting at 40% as detected using asenun bactericidal assay;

FIG. 13 illustrates Geometric mean bacteremia in the blood of groups of5 day old infant rats 24 h post-infection with 1×10⁸ cfu/ml galE mutantgiven simultaneously with: (i) no antibody; (ii) MAb B5 (10 μg dose);(iii) MAb B5 (100 μg dose); or (iv) MAb 735, a positive controlanti-capsular antibody (2 μg dose);

FIG. 14 illustrates a Western blot showing purified LPS from Neisseriameningitidis MC58 and galE mutant probed with MAb B5 (ascites fluid1:2000) detected using anti-mouse IgG alkaline phosphatase and BCIP/NBTsubstrate; and

FIG. 15 illustrates a FACS profile comparing surface labeling of liveNeisseria meningitidis MC58 and galE mutant (5×108 org./ml) with MAb B5(culture supernatant 1:50) detected using anti-mouse IgG (FITClabelled).

EXAMPLES OF THE INVENTION Example 1 Identification of ImmunogenicEpitopes in Neisseria Meningitidis Introduction

We investigated the conservation and antibody accessibility of innercore epitopes of Neisseria meningitidis lipopolysaccharide (LPS) becauseof their potential as vaccine candidates. An IgG3 murine monoclonalantibody (MAb), designated MAb B5, was obtained by immunizing mice witha galE mutant of Neisseria meningitidis H44/76 (B.15.P.1.7.16 immunotypeL3). We have shown that MAb B5 can bind to the core LPS of wild-typeencapsulated MC58 (B.15.PI.7.16 immunotype L3) organisms in vitro andex-vivo. An inner core structure recognized by MAb B5 is conserved andaccessible in 26/34 (76%) of Group B and 78/112 (70%) of Groups A, C, W,X, Y and Z strains. Neisseria meningitidis strains which possess thisepitope are immunotypes in which phosphoethanolamine (PEtn) is linked tothe 3-position of the P-chain heptose (HepII) of the inner core. Incontrast, Neisseria meningitidis strains lacking reactivity with MAb B5have an alternative core structure in which PEtn is linked to anexocyclic position (i.e. position 6 or 7) of HepII (immunotypes L2, L4and L6) or is absent (immunotype L5). We conclude that MAb B5 definesone or more of the major inner core glycoforms of Neisseria meningitidisLPS.

These findings encourage the possibility that immunogens capable ofeliciting functional antibodies specific to inner core structures couldbe the basis of a vaccine against invasive infections caused byNeisseria meningitidis.

In summary, we report that a monoclonal antibody, designated B5, hasidentified a crossreacting epitope on the LPS of the majority ofnaturally occurring, but genetically diverse strains of Neisseriameningitidis. Critical to the epitope of strains recognized by themonoclonal antibody B5 is a phosphoethanolamine (PEtn) on the 3-positionof the P-chain heptose (HepII) (FIG. 1). In contrast, all Neisseriameningitidis strains lacking reactivity with MAb B5 are immunotypescharacterized by the absence of PEtn substitution or by PEtnsubstitution at an exocyclic position (i.e. position 6 or 7) of HepII(FIG. 1). Thus, a limited repertoire of inner core LPS variants is foundamong natural isolates of Neisseria meningitidis strains and thesefindings encourage the possibility that a vaccine might be developedcontaining a few glycoforms representative of all natural Neisseriameningitidis strains.

Materials and Methods Bacterial Strains

The Neisseria meningitidis strains MC58 and H44/76 (both B:15:P1.7.16immunotype L3) have been described previously (Virji, M., H. Kayhty, D.J. P. Ferguson, J. E. Heckels, and E. R. Moxon, 1991. Mol Microbiol 5:1831-1841, Holten, E. 1979. J Clin Microbiol 9: 186-188). Derivatives ofMC58 and H44/76 with defined alterations in LPS were obtained byinactivating the genes, galE (Jennings, M. P., P. van der Ley, K. E.Wilks, D. J. Maskell, J. T. Poolman, and E. R. Moxon. 1993. MolMicrobiol 10: 361-369), Isi (Jennings, M. P., M. Bisercic, K. L. Dunn,M. Virji, A. Martin, K. E. Wilks, J. C. Richards, and E. R. Moxon. 1995.Microb Pathog 19: 391-407), IgtA, IgtB (Jennings, M. P., D. W Hood, I.R. Peak, M. Virji, and E. R. Moxon. 1995. Mol Microbiol 18: 729-740)rfaC (Stoiljkovic, I., V. Hwa, J. Larson, L. Lin, M. So, and X. Nassif.1997. FEMS Microbiol Lett 151: 41-49), icsA and icsB (van der Ley, P.,M. Kramer, A. Martin, J. C. Richards, and J. T. Poolman, 1997. FEMSMicrobiol Len 146: 247-253) (Table 1). Other wild type Neisseriameningitidis strains used in the study were from three collections: 1)representatives of immunotypes L1-L12 (Poolman, J. T., C. T. P. Hopman,and H. C. Zanen. 1982. FEMS Microbiol Lett 13: 339-348); 2) globalcollection of 34 representative Neisseria meningitidis Group B strains(Seiler, A., R. Reinhardt, J. Sakari, D. A. Caugant, and M. Achtman.1996. Mol Microbiol 19: 841-856); 3) global collection of 100 strainsfrom 107 representative Neisseria meningitidis strains of all majorserogroups (A, B, C, W, X, Y, Z) (Maiden, M. C. J., J. A. Bygraves, E.Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth,D. A. Caugant, I. M. Feavers, M. Achtman, and G. B. Spratt. 1998 PNAS95: 3140-3145).

Capsule deficient and galE mutants were constructed in six Neisseriameningitidis Group B strains obtained from the collection as describedin (Seiler, A., et al., 1996. Mol Microbiol 19: 841-856) (Table 1).Other related Neisseria strains studied included 10 strains of Neisseriagonorrhoeae and commensal strains lactamica (8 strains), polysaccharea(1 strain), mucosa (1 strain), cinerea (1 strain), elongata (1 strain),sicca (1 strain) and subflava (1 strain). Other Gram negative organismsincluded: Haemophilus influenzae type b (7 strains), Haemophilus somnus(1 strain), non-typable Haemophilus influenzae (8 strains), Escherichiacoli (1 main) and Salmonella typhimurium (1 strain) and its isogenic LPSmutants (rfaC, rfaP, rfaI) (Table 1).

Bacterial Culture In Vitro

All strains were grown overnight at 37° C. on standard BHI medium base(Oxoid) in an atmosphere of 5% C0₂.

Bacterial Culture In Vivo Using the Chick Embryo Model

To determine the accessibility of inner core epitopes of Neisseriameningitidis grown in vivo the chick embryo model was used (Buddingh, G.J., and A. Polk. 1937. Science 86: 20-21, Buddingh, G. J., and A. Polk.1939. J Exp Med 70: 485-498, Schroten, H., M. Deadman, and E. R. Moxon.1995. Pediar. Grenzgeb. 34: 319-324). The method was modified using aninoculum of 10⁴ and 10⁵ Neisseria meningitidis organisms in a finalvolume of 0.1 ml, to infect the chorio-allantoic fluid of 10 day oldPure Sussex chick eggs (obtained from the Poultry Unit Institute ofAnimal Health, Compton, Berks). After overnight incubation (37° C.) theallantoic fluid (approx. 3-5 mls) was removed from the eggs and thebacteria recovered after centrifugation at 350×g for 15 minutes. Theorganisms were washed in sterile phosphate buffered saline (PBS) andstored in Greaves solution (5% BSA, 5% Sodium Glutamate, 10% Glycerol)at −70%° C.

LPS Extraction

LPS samples were obtained from an overnight growth of Neisseriameningitidis plated on 5 BHI plates from which the organisms werescraped and suspended in 30 ml 0.05% phenol in PBS and incubated at roomtemperature for 30 minutes. Alternatively, batch cultures were preparedin fermenters using bacteria from an overnight growth (6 plates) in 50ml Difco Bacto Todd Hewitt broth (Difco) to inoculate 2.5 L of the samemedium. For insertion mutant strains, the medium contained 50 μg/mlkanamycin. Following incubation at 37° C. for 6-8 h the culture wasinoculated into 60 L of Bacto Todd Hewitt broth in a New BrunswickScientific 1F-75 fermenter. After overnight growth (17 h at 37° C.), theculture was killed by addition of phenol (1%), and chilled to 15° C. andthe bacteria were harvested by centrifugation (13,000 g for 20 min)(Wakarchuk W., et al., 1996. J. Biol. Chem. 271: 19166-19173). In eithercase, the crude LPS were extracted from the bacterial pellet using thestandard hot phenol-water method (Westphal, O., and J. K. Jann. 1965.Meth. Carbohydr. Chem. 5:83-91) and purified from the aqueous phase byrepeated ultracentrifugation (105,000×g, 4° C., 2×5 h) (Masoud, H., E.R. Moxon, A. Martin, D. Krajcarski, and J. C. Richards. 1997.Biochemistry 36: 2091-2103).

Tricine Gels

Equivalent amounts of whole-cell lysates of Neisseria meningitidisstrains or purified LPS were boiled in dissociation buffer and separatedon standard tricine gels (30 mA for 18 h) (Lesse, A. J., A. A.Campagnari, W. E. Bittner, and M. A. Apicella. 1990. J Immunol Methods126: 109-117). Gels were fixed and silver-stained as per manufacturer'sinstructions (BioRad). To determine the presence of sialic acid, wholecell lysates were incubated with 2.5 μl neuraminidase at 37° C. for18-20 h (4 U/ml Boehringer 1585886) and then with 5 μl proteinase K at60° C. for 2-3 h to remove proteins (Boehringer 1373196) prior toseparation on tricine gels (16.5%).

Characterization of LPS from MAb B5 Negative Strains

LPS from wild-type and galE, cap-mutant MAb B5 negative strains wereO-deacylated with anhydrous hydrazine as described previously (Masoud,H., E. R. Moxon, A. Martin, D. Krajcarski, and J. C. Richards. 1997.Biochemistry 36: 2091-2103). O-deacylated LPS were analyzed byelectrospray mass spectrometry (ES-MS) in the negative ion mode on a VGQuattro (Fisons Instruments) or API 300 (Perkin-Elmer/Sciex) triplequadruple mass spectrometer. Samples were dissolved in water which wasdiluted by 50% with acetonitrile:water:methanol:1% ammonia (4:4:1:1) andthe mixture was enhanced by direct infusion at 4 μl/min. Deacylated anddephosphorylated LPS (L8 odA HF) was prepared according to the followingprocedure. LPS (160 mg) was treated with anhydrous hydrazine (1.5 ml)with stirring at 37° C. for 30 minutes. The reaction was cooled (0° C.),cold acetone (−70° C., 50 ml) was added gradually to destroy excesshydrazine, and precipitated O-deacylated LPS (L8 odA) was obtained bycentrifugation. L8 odA was washed twice with cold acetone, andredissolved in water and lyophilized. The structure of L8 odA wasconfirmed by negative ion ES-MS before proceeding to dephosphorylation.L8 odA was dephosphorylated by treatment with 48% aqueous hydrogenfluoride (10 ml) at 0° C. for 48 h. The product was dialyzed againstwater, and the O-deacylated, dephosphorylated LPS sample (L8 odA HF) waslyophilized (50 mg). Loss of phosphate was confirmed by ES-MS.

Molecular Modeling

Molecular modeling of LPS epitopes was carried out as describedpreviously by Brisson, J. R., S. Uhrinova, R. J. Woods, M. van der Zwan,H. C. Jarrell. L. C. Paoletti, D. L. Kasper, and H. Jennings. 1997.Biochemistry 36: 3278-3292). The starting geometry for all sugars wassubmitted to a complete refinement of bond lengths, valence and torsionangles by using the molecular mechanics program MM3(92) (QPCE). Allcalculations were performed using the minimized co-ordinates for themethyl glycoside. The phosphorus groups were generated from standardco-ordinates (Alchemy, Tripos software) and minimum energy conformationsfound in crystal structures. Calculations were performed using theMetropolis Monte Carlo (MMC) method. All pendant groups were treated asinvariant except for the phosphorus groups which were allowed to rotateabout the Cx-Ox and Ox-P bonds. The starting angles for theoligosaccharide were taken from the minimum energy conformers calculatedfor each disaccharide unit present in the molecule. 24-dimensional MMCcalculations of the hexasaccharides with or without PEtn groups attachedwere carried out with 5000 macro moves. The graphics were generatedusing the Schakal software (Egbert Keller, KristallographischesInstitutder Universität, Freibury, Germany).

Antibodies Rabbit Polyclonal Antibody

We used a rabbit polyclonal antibody specific for Group B Neisseriameningitidis capsular polysaccharide obtained by immunizing a rabbit sixtimes sub-cutaneously with lysates of MC58 at 2-week intervals. Thefirst and second immunizations contained Freund's complete adjuvant andFreund's incomplete adjuvant respectively. Serum was obtained from bleed6. To increase specificity for the Group B capsular polysaccharide,rabbit polyclonal antibody (1 ml) was incubated overnight at 4° C. withethanol-fixed capsule-deficient MC58 (5×10⁹ org./ml). This pre-adsorbedpolyclonal antibody did not react with a capsule-deficient mutant ofMC58 using immunofluorescence microscopy.

Monoclonal Antibodies to Inner Core LPS

Murine monoclonal antibodies to H44/76 galE LPS were prepared bystandard methods. Briefly, 6-8 week old Balb/c mice were immunized threetimes intraperitoneally followed by one intravenous injection withformalin-killed galE mutant whole cells. Hybridomas were prepared byfusion of spleen cells with SP2/O-Ag 14 (Shulman, M., C. D. Wilde, andG. Kohler. 1978. Nature 276: 269-270) as described (Carlin, N. I., M. A.Gidney, A. A. Lindberg, and D. R. Bundle. 1986. J Immunol 137:2361-2366). Putative hybridomas secreting galE specific antibodies wereselected by ELISA employing purified LPS from L3 and its galE mutant andL2. Ig class, subclass and light chain were determined by using anisotyping kit (Amersham Canada Ltd, Oakville, Ontario). Clones wereexpanded in Balb/c mice following treatment with pristane to generateascitic fluid. Spent culture supernatant was collected following invitro culture of hybridoma cell lines. Further testing of galE MAbs wascarried out by screening against purified LPS from Neisseriameningitidis L3 IgtA, IgtB and IgtE mutant strains (FIG. 1), andSalmonella typhimurium Ra and Re mutants. One of the MAbs, MAb B5(IgG₃), was selected for more detailed study.

Immunotyping Monoclonal Antibodies

To determine the immunotypes of Neisseria meningitidis Strains studies,especially L2 and L4-L6, the following murine MAbs were used in dotblots and whole cell ELISA:MN42F12.32 (L2, 5), MN4A8B2 (L3, 7, 9),MN4C1B (L4, 6, 9), MN40G11.7 (L6), MN3A8C (L5) (Scholten, R. J., et al.,J Med Microbial. 41: 236-243).

Human Umbilical Vein Endothelial Cell (HUVEC) Assay

Cultured human umbilical vein endothelial cells (HUVECs) were preparedas described previously (Virji, M., et al., 1991. Microb Pathog 10:231-245) and were infected with strains of Neisseria meningitidis for 3h at 37° C. Neisseria meningitidis strains were grown wither in vitro orin vivo using the chick embryo model (as described above). Theaccessibility of the inner core LPS epitopes of whole-cell Neisseriameningitidis to specific MAb B5 was determined using immunofluorescenceand confocal microscopy. Gelatin-coated glass coverslips coated withHUVECs were infected with wild-type Neisseria meningitidis as describedpreviously (Virji, M., et al., 1991. Mol Microbial. 5: 1831-1841),except bacteria were fixed with 0.5% paraformaldehyde for 20 min insteadof methanol. For accessibility studies, coverslips were washed with PBS,blocked in 3% BSA-PBS and incubated with MAb B5 culture supernatant andpre-adsorbed polyclonal rabbit anti-capsular antibody. Binding ofantibody to wild-type Neisseria meningitidis strains was detected byanti-mouse IgG rhodamine (TRITC) (Dako) and anti-rabbit IgG fluorescein(FITC) (Sigma). HUVECs were stained using diaminophenylamine DAPI (1μg/ml) (Sigma). Mounted coverslips were viewed for immunofluorescenceusing appropriate filters (Zeiss Microscope with Fluorograbber, AdobePhotoshop or confocal microscope (Nikon Model).

ELISA Purified LPS ELISA

A solid phase indirect ELISA employing purified LPS was used todetermine the binding specificities of MAbs. Nunc maxisorp plates werecoated overnight with 1.0 μg/well of purified LPS derived from wild typeand mutants. LPS (10 μg/ml) was diluted in 0.05 M carbonate buffercontaining 0.02 M MgCl₂, pH 9.8. Non-specific binding sites were blockedfor 1 h with 1% BSA-PBS (Sigma) and washed three times with PBS Tween 20(0.05% v/v) (PBS-T). Plates were incubated for 1 h with MAb B5 culturesupernatant and washed three times in PBS-T. Primary antibody wasdetected using anti-mouse IgG-alkaline phosphatase (Sigma: CedarlaneLaboratories Ltd.) incubated for 1 h, washed three times in PBS-T, anddetected using p-nitrophenyl phosphate AP substrate system (Sigma:Kirkegaard & Perry Laboratories). The reaction was stopped after 1 hwith 50 μl 3 M NaOH and absorbance determined at OD A_(405-410nm)(Dynatech EIA plate reader).

Inhibition ELISA

For inhibition ELISA studies, MAb B5 was incubated with purified LPSsamples prior to addition to L3 galE LPS coated plates and assayed asdescribed above.

Whole Cell ELISA

Whole cell (WC) ELISA was performed using heat-inactivated lysates ofNeisseria meningitidis organisms as described previously (Abdillahi, H.,and J. T. Poolman. 1988. J Med. Microbial. 26:177-180). Nunc Maxisorp96-well plates were coated with 100 μl bacterial suspension (OD of 0.1at A_(820nm)) overnight at 37° C., blocked with 1% BSA-PBS and identicalprotocol followed as for LPS ELISA.

Dot Blots

Bacterial suspensions prepared as above (2 μl) were applied to anitrocellulose filter (45 micron, Schleicher and Schueller) and allowedto air dry. The same procedure as described for WC ELISA was followedexcept the detection substrate was 5-bromo-4-chloro-3-indoylphosphate/nitroblue-tetrazolium (BCIP/NBT) (2 mg/ml; Sigma). The colourreaction was stopped after 30 min by several washes with PBS and blotswere air-dried.

Results

To investigate the potential of inner core LPS structures of Neisseriameningitidis as vaccines, we have studied the reactivity of an isotypeIgG₃ murine monoclonal antibody (MAb), designated B5, raised againstNeisseria meningitidis stain H44/76 immunotype L3 galE mutant. MAb B5was one of seven monoclonal antibodies to LPS inner core producedagainst Neisseria meningitidis immunotype L3 galE by standardimmunological methods (see Methods). Preliminary ELISA testing showed B5cross-reacted with LPS from L3 parent strain and with galE (IgtE), IgtAand IgtB mutants, but did not cross-react with Salmonella typhimurium Raor Re LPS.

In order to determine the specific inner core epitope recognized by MAbB5, various Neisseria meningitidis strains of known structure wereexamined in ELISA for cross reactivity (FIG. 2). The most significantfinding of this analysis was that Neisseria meningitidis immunotype L4LPS was not recognized by MAb B5. The only structural difference betweenimmunotypes L4 and L3 (which is recognized by MAb B5) is the position ofattachment of the PEtn group (FIG. 3). In immunotype L3 LPS the PEtn isattached at the 3-position of HepII, whereas in immunotype L4 LPS thePEtn is attached at the 6- or 7-position of HepII (FIG. 3).Additionally, LPS from immunotype L2 and its galE mutant (in which thePEtn group is attached at the 6-position and a glucose residue ispresent at the 3-position of HepII) are not recognized by MAb B5.Immunotype L5, which has no PEtn in the inner core, is not recognized byB5, whereas immunotype L8 and its galE mutant which have PEtn at the3-position of HepII are recognized. These results suggest that MAb B5specifically recognizes PEtn when it is attached at the 3-position ofHepII.

In order to prove the essential inclusion of PEtn in the epitoperecognized by MAb B5, immunotype L8 O-deacylated (odA) LPS wasdephosphorylated (48% HF, 4° C. 48 h) (FIG. 3). The absence of PEtnfollowing dephosphorylation was confumed by ES-MS analysis. As indicatedin FIG. 4, dephosphorylation of L8 odA LPS abolished reactivity to MAbB5. To further characterize the epitope recognized by MAb B5, severalstructurally defined genetic mutants of immunotype L3 were screened forcross-reactivity (FIG. 4). The highly truncated LPS of mutant strainicsB were only weakly recognised, while mutant strain icsA LPS was notrecognised by MAb B5. These results suggest that the presence of glucoseon the proximal heptose reside (HepI) is not absolutely necessary forbinding by B5 but is required for optimal recognition (FIG. 1).Furthermore, MAb B5 does not bind LPS in which both the glucose on theα-chain, HepI, and the N-acetylglucosamine residue on the β chain,HepII, are absent. This suggests that the presence ofN-acetylglucosamine is required to present the PEtn residue in thecorrect conformation for binding by MAb B5. Genetic modifications thatproduce severely truncated LPS glycoforms were also examined forreactivity with MAb B5. LPS from immunotype L3 Isi which has atrisaccharide of Hep-Kdo-Kdo attached to lipid A, and L3 PB4 which onlycontains the Kdo disaccharide and lipid A were not recognized by MAb B5(FIG. 4). Inhibition ELISA studies (data not shown) were in accord withthis result, thus confirming the specificity of MAb B5 to the PEtnmolecule linked at the 3-position of HepII.

To demonstrate the ability of MAb B5 to recognize this inner coreepitope in encapsulated strains, we devised an assay in which naturalisolates of Neisseria meningitidis were studied when they were grown onand became adherent to tissue cultured cells (HUVECs). Initially, thismethodology was developed using the fully encapsulated strain MC58. Theadvantages of using the HUVEC assay were that they provided a monolayerof endothelial cells to which the bacteria could adhere and that theyprovided a biologically relevant environment. Previous attempts usingNeisseria meningitidis directly adherent to gelatin- or matrigel-coatedcoverslips resulted in low numbers of adherent bacteria after repeatedwashings and high nonspecific background staining.

Primary antibodies, MAb B5 and a polyclonal anti-capsular antibody weredetected by anti-mouse TRITC and anti-rabbit FITC respectively. Thisdemonstrated that an inner core LPS epitope of the fully encapsulatedstrain (MC58) was accessible to MAb B5 (FIG. 5 a). Confocal microscopyshowed that MAb B5 and anti-capsular antibodies co-localised. Inaddition to this in vitro demonstration of accessibility of MAb B5 toinner core LPS, we also investigated organisms grown in vivo using thechick embryo model. Strain MC58 (10⁴ org./ml) was inoculated intochorio-allantoic fluid of 10 day old chick embryos and harvested thenext day to provide ex-vivo organisms. The results of confocalmicroscopy were identical to those observed in vitro, that is MAb B5 andanti-capsular antibodies co-localised (FIG. 5 b). This demonstrated thatthe inner core LPS epitopes were also accessible in vivo onwhole-encapsulated wild-type Neisseria meningitidis.

The observation of double staining of the inner core LPS epitope in thepresence of capsule is key to the concept of this approach and thereforea number of controls were used to confirm the validity finding. Theseincluded: (i) double staining a MAb B5 negative e.g. immunotype L4strain with MAb B5 and anti-capsular antibody. This resulted in noreactivity of MAb B5 on rhodamine filter but positive reactivity withanti-capsular antibody. This rules out a band passing effect during therecording of the pictures; (ii) single staining of encapsulated MAb B5positive strains with either MAb B5 alone or anti-capsular antibodyalone followed by staining with rhodamine or FITC, respectively. Whenviewed on the appropriate wavelength there was no cross-reactivityduring immunofluorescent staining nor any band-passing effect; (iii)double-staining of a MAb B5 positive or negative strain without capsulewith MAb B5 and anti-capsular antibody resulted in no capsular stainingbut either MAb B5 positive or negative reactivity when viewed on therhodamine filter. This excluded cross-reactivity during staining orband-passing effect resulting in artefactual inner core staining.

To survey the extent of MAb B5 reactivity with other Neisseriameningitidis strains, three collections were investigated.

i) 12 strains representative of LPS immunotypes L1-L12

ii) 34 Group B strains selected to represent genetically diverseisolates from many different countries obtained between the years1940-1988 (Seiler, A., R. Reinhardt, J. Sakari, D. A. Caugant and M.Achtman. 1996. Mol Microbiol 19: 841-856)

iii) a global collection of 107 genetically diverse strains representingall capsular serogroups, also obtained from different countries from1940-1994 (Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E.Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M.Feavers, M. Achtman, and G. B. Spratt. 1998. PNAS 95: 3140-3145).

Of the 12 immunotypes, MAb B5 recognized the LPS of strains in which theinner core oligosaccharide has a PEtn linked to the 3-position of HepII(Table 2 and FIG. 1). Thus, immunotypes L2, L4, L6 did not react withMAb B5, whereas immunotypes L1, L3, L7-L12 were recognized by MAb B5.This confirmed that the presence of PEtn in the 3 position of the HepIIis necessary to confer MAb B5 reactivity (FIG. 3).

To investigate further the MAb B5 reactivity with other Group B strains,a collection of genetically diverse strains was studied (Seiler, A., andR. Reinhardt, J. Sakari, D. A. Caugant, and M. Achtman. 1996. MolMicrobiol 19: 841-856). MAb B5 reactivity was detected in 26/34 (76%) ofGroup B Neisseria meningitidis stains tested. This includedrepresentative strains of ET-5, ET-37, A4 and Lineage-3. This representsthe most complete available collection of hyper-invasive lineages ofNeisseria meningitidis Group B strains.

We obtained capsule-deficient and galE mutants from six of eight of theMAb B5 negative Group B strains (transformations were unsuccessful inthe other two strains). Theses were also negative with MAb B5 using dotblot, whole cell ELISA or immunofluorescence, with the exception of aBZ157 galE cap-mutant; which had low level reactivity both byimmunofluorescence and dot blot. The MAb B5 strains were characterizedusing a battery of immunotyping MAbs. We determined the immunotype ofthe eight MAb B5 negative strains using combinations of the appropriateMAbs (see Methods) and dot blots of WC lysates (obtained from Peter vander Ley) (Table 3). In addition, structural fingerprinting of the innercore region of MAb B5 negative strains was performed by ES-MS onO-deacylated LPS from five of the respective capsule-deficient galEmutants (1000, NGE30, EG327, BZ157, NGH38) (Table 4). Strains 1000,NGE30, EG327 were non-typical by MAbs and LPS from these strains lackedPEtn on HepII of the inner core. BZ157, which corresponded to immunotypeL2 by MAbs contained PEtn in the inner core, and by analogy to L2 at the6/7 position of HepII (Table 3). NGH38 was immunotype L2, L5 andanalogous to L2 by structural analysis. Those strains that werenon-typable failed to react with MAbs that recognize L3, 7, 9, L6, L2,5, L4, 6, 9. However, 15/17 MAb B5 negative Neisseria meningitidisstrains (all serogroups) were positive for L2, 5 and all MAb B5 positivestrains were positive for L3, 7, 9. No reaction with any immunotypingMAbs was observed with 8/32 MAb B5 negative strains and 24/68 of MAb B5positive strains.

To determine if the degree of sialylation of the LPS was a factor in theability of MAb B5 to recognize its inner-core epitope, MAb B5 negativestrains were examined by LPS gels. MAb B5 reactivity was unaffected byvarying the state of sialylation through exposure to neurominidase asdescribed in methods (FIG. 6). Furthermore, strain MC58, with which theMAb B5 reacted strongly, was found to be highly sialylated (FIG. 6) andthis was confirmed by ES-MS of purified O-deacylated LPS (data notshown). Therefore our data did not support a contribution of sialylationto the lack of MAb B5 reactivity.

With respect to the other Neisseria species, MAb B5 also recognized theinner core LPS of five strains of Neisseria gonorrhoeae (F62, MS11,FA19, 179008, 150002) (two were negative) and (at least) two strains ofNeisseria lactamica (L19, L22). However, MAb B5 did not react with onestrain each of Neisseria polysaccharide (M7), Neisseria mucosa (F1),Neisseria cinerea (Griffiss, J. M., J. P. O'Brien, R. Yamensaki, G. D.Williams, P. A. Rice, and H. Schneider. 1987. Infect Immun55:1792-1800), Neisseria elongata (Q29), Neisseria sicca (Q39) andNeisseria subflava (U37). Also MAb B5 did not react with Escherichiacoli (DH5 alpha), Salmonella typhimurium (LT2) or its isogenic LPSmutants (rfaC, rfaI, rfaP).

Finally, we investigated the reactivity of MAb B5 with 100 strains thatincluded representatives of serogroups A, B, C, W, X, Y and Z (Maiden,M. C. J., et al., 1998. PNAS 95: 3140-3145). Of these strains, 70% wereMAb B5 positive. Clustering according to genetic relatedness wasevident. For example, none of the MAb B5 negative stains were in the ET5complex. Among Group A strains, MAb B5 positive and negative stains alsofell into distinct clusters. For example, lineages I-III and lineage A4were positive and lineage N−1 was negative. This collection, togetherwith that described in (Seiler, A., et al., 1996. Mol Microbiol 19:841-856) represents the most complete set available for knownhyperinvasive lineages in all major serogroups of Neisseria meningitidisstrains.

Discussion and Conclusions

The pre-requisites for any candidate Neisseria meningitidis Group Bvaccine would be that it contains a highly conserved epitope(s) that isfound in all Group B stains and is accessible to antibodies in thepresence of capsule. Our approach has combined genetics, structuralanalysis and immunobiology to define candidate epitopes in inner coreLPS of Neisseria meningitidis Group B. This study uses murine MAb B5,isotype IgG3, which was raised to a genetically defined immunotype L3galE mutant in order to specifically target inner-core LPS epitopes. Theepitope(s) recognized by MAb B5 was defined by cross-reactivity studieswith purified LPS glycoforms of known structure. MAb B5 recognized allLPS glycoforms in which the PEtn is at the 3-position of HepII(immunotypes L1, L3, L7, L8 and L9) and failed to react with immunotypeswhere PEtn is at the 6- or 7-position (L2, L4 and L6) or absent fromHepII (L5) (FIG. 1). MAb B5 reacted with 70% Neisseria meningitidisstrains tested from the two most complete sets of Neisseria meningitidisstrains available word-wide (Seiler, A., et al., 1996. Mol Microbiol 19:841-856, 35). Of these strains, 76% of Neisseria meningitidis Group Bstrains tested were positive with MAb B5 and 70% of a collection thatincluded all Neisseria meningitidis serogroups tested was positive withMAb B5. Therefore, it may be envisaged that a vaccine containing alimited number of glycoforms, representing all the possible PEtnpositions (none, 3 and 6/7) on HepII on the inner core, would cover 100%of Neisseria meningitidis Group B strains.

The LPS structures of MAb B5 negative strains were confirmed bystructural analysis. Two structural variants were recognised. Onevariant without PEtn in the inner core LPS (e.g. NGE30, EG327, 1000);and the other, with PEtn group of HepII (e.g. BZ157, NGH38) at the 6- or7-position instead of the 3-position.

With a view to developing inner core LPS epitopes as vaccine candidates,it is significant that there were no effects of the capsule on MAb B5accessibility, as shown by co-localization of the anti-capsule antibodyand MAb B5 in wild-type organisms (MC58) grown in vitro and in vivo byconfocal microscopy (FIGS. 5 a and b). Nor did the presence or absenceof sialic acid have an effect since both MAb B5 positive and negativestrains had high sialylation states as shown by tricine gels (FIG. 6)and confirmed by ES-MS (data not shown).

There was no evidence of phase variation in MAb B5 positive or negativestrains in this study, with the exception of one strain (BZ157) whichhad a very low level of MAb B5 positive strains in parent and galEmutant (0.06%) (data not shown). Structural analysis of LPS extractedfrom these two variants is currently under investigation.

Three dimensional space filling models of the inner core LPS of L3 andL4 immunotypes show that the position of the PEtn, either 3- or6-position respectively (shown in brown), alters the accessibility andconformation of PEtn in the inner core epitope (FIG. 3). The moststriking example of the importance of PEtn for MAb B5 reactivity wasobserved when PEtn was removed from the immunotype L8 (MAb B5 positive)by treatment with hydrogen fluoride (HF) which totally abolished MAb B5reactivity.

Previous studies with oligosaccharide conjugates in mice and rabbitshave demonstrated that PEtn is important in immunogenicity andfunctional activity of polyclonal antibodies (Verheul, A. F., et al.,1991. Med Immun 59: 843-851). These studies identified two sets ofpolyclonal antibodies. One set resulting from L1 and L3, 7, 9oligosaccharides had PEtn in the 3-position of HepII, were immunogenic,had opsonophagocytic (OP) and chemiluminescence in oxidative burstreaction, but had no serum bactericidal activity. The other set ofantibodies resulting from L2 conjugates (6- or 7-position or withoutPEtn at HepII) were poorly immunogenic and had greatly reduced OPactivity and chemiluminescence (Verheul. A. F., A. K. Braat, J. M.Leenhouts, P. Hoogerhout, J. T. Poolman, H. Snippe, and J. Verhoef.1991. Infect Immun 59: 843-851). Future studies will look at the safetyand immunogenicity of inner core LPS-conjugates (PEtn at 3-position ofHepII and alternative glycoforms) and the functional ability of thesepolyclonal antibodies in opsonic and serum bacterial assays, initiallyin mice and rabbits. Preliminary studies using MAb B5 in anopsonophagocytosis assays with Neisseria meningitidis strain MC58 anddonor human polymorphonuclear cells suggest MAb B5 is opsonic in thepresence of complement and that the uptake of Neisseria meningitidisbacteria correlates with an oxidative burst reaction within theneutrophil. MAb B5 does not appear to have any significant serumbactericidal activity with Neisseria meningitidis strain MC58, howeverthis is not unexpected in view of its isotype (IgG3). The functionalityof MAb B5 is currently under further investigation.

In conclusion, MAb B5 recognizes a conserved inner core epitope in whichthe PEtn is at the 3-position of HepII. This epitope was present in 76%Neisseria meningitidis Group B strains and 70% of all Neisseriameningitidis serogroups, and was accessible in the presence of capsule.A limited number of alternative glycoforms have been identified that arenot recognized by MAb B5 where the PEtn is either absent or at anexocyclic position of HepII. Therefore, a vaccine containing a limitednumber of glycoforms might give 100% coverage of all Neisseriameningitidis Group B strains.

TABLE 1 Bacterial strains. Relevant immunotype (bold) and Species Straingenotype(italics) Source/reference Neisseria meningitidis MC58 L3 Virji,M., H. Kayhyt, D. J. P., CSF isolate Ferguson, J. E. Heckels, and E. R.Moxon. 1991. Mol Microbiol5: 1831-1841 H44/76 L3 Holton, E. 1979. J ClinMicrobiol 9: 186-188 MC58 galE Jennings, M. P., P. van der Ley, K. E.Wilks, D. J. Maskell, J. T. Poolman, and E. R. Moxon. 1993. MolMicrobiol10: 361-369 MC58 Isil(rfaF Jennings, M. P., M. Bisercic, K. L. Dunn, M.Virji, A. Martin, K. E. Wilks, J. C. Richards, and E. R. Moxon. 1995Microb Pathog19: 391-407 MC58 IgtA Jennings, M. P., D. W. Hood, I. R.Peak, M. Viji, and E. R. Moxon. 1995. Mol Microbiol 18: 729-740 MC58IgtB Jennings, M. P., D. W. Hood, I. R. Peak, M. Viji, and E. R. Moxon.1995. Mol Microbiol 18: 729-740 H44/76 rfaC Stolljokovic, I., V. Hwa, J.Larson, L. Lin, M. So, and X. Nassif. 1997. FEMS Microbial. Lett 15 1:41-49 H44/76 icsA van der Ley, P., M. Kramer. A. Martin, J. C. Richards,and J. T. Poolman. 1997. FEMS Microbiol. Lett 146: 247-253 126E L1-L12Poolman, J. T., C. T. P. Hopman, 35E; H44/76; 89I; M981 RESPECTIVELY andH. C. Zanen. 1982. FEMS Microbial. Lett 13: 339-348 M992 6155; 892257;M978; 120M; 7880; 7889; 3200 BZ157 L2 Seiler, A., R Reinhart, J. Sakari,D. A. Caugant, and M. Achhnan. 1996. Mol Microbiol 19: 841-856 BZ157galE This study 1000 NT Seiler, A., R Reinhart, J. Sakari, D. A.Caugant, and M. Achhnan. 1996. Mol Microbiol 19: 841-856 1000 galE Thisstudy NGE30 NT Seiler, A., R Reinhart, J. Sakari, D. A. Caugant, and M.Achhnan. 1996. Mol Microbiol 19: 841-856 NGE30 galE This study EG327 NTSeiler, A., R Reinhart, J. Sakari, D. A. Caugant, and M. Achhnan. 1996.Mol Microbiol 19: 841-856 EG327 galE This study NGH38 L2, 5 Seiler, A.,R Reinhart, J. Sakari, D. A. Caugant, and M. Achhnan. 1996. MolMicrobiol 19: 841-856 EG328 NT Seiler, A., R Reinhart, J. Sakari, D. A.Caugant, and M. Achhnan. 1996. Mol Microbiol 19: 841-856 EG328 galE Thisstudy 3906; NGH15; Seiler, A., R Reinhart, J. BZ133; BZ83; EG329;Sakari, D. A. Caugant, and M. SWZ107; BZ198; NGH4 Achhnan. 1996. MolMicrobiol 1; NG4/88; 2970; BZ147; 19: 841-856 NGG40; NGH36; NG3/88;NGF26; NG6/33; NGH38; NGE28; BZ169; 528; DK353; BZ232 DK24; BZ159; BZ10;BZ163; NGP20 B40; Z4024; Z4081; Z2491; Z3524; (35) Z3906; Z5826; BZ10;BZ163; B6116/77; L93/4286; NG3/88; NG6/88; NGF26; NGE31; DK24; 3906;EG328; EG327; 1000; B534; A22; 71/94; 860060; NGG40; NGE28; NGH41;890326; 860800; NG4/88; E32; 44/76; 204/92; BZ8; SWZ107; NGH38; DK353;BZ232; E26; 400; BZ198; 91/40; NGH15; NGE30; 50/94 88/03415; NGH36;BZ147; 297-0 Neisseria lactamica Brian Spratt & Noel (L12. LI3. LI 7,L18, L19, L20, L22) polysaccharea (P4), mucosa (M7), cinerea (Fl).elongata (18), sicca (Q29), subflova (U37) Neisseria gonorrhoeae: F62,MS11, FA19, FA1090, 179008, R. Goldstein 150002, 15253 SN-4 StaffanNormavk P9-2 M. Virji Haemophilus injluenzae Hood, D. W., M. E. Deadman,T. type b Allen, H. Masoud, A. Martin, J. R. Eagan; 7004; Rd5B33; opsxBrisson, R. Fleischman, J. C. 3Fe; E3Fi; E1B1 rfaF Venter, J. C.Richards, and E. R. orfH-l. Moxon. 1996. Mol Microbiol 22: 951-964 IpxAPLAK33 Steeghs, L., R den Hartog, A. denBoer, B. Zomer, P. Roholl, andP. van der Ley. 1998. Nature 392: 449-450. Haemophilus somnus J.Richards 738 L1 Non-typable J. Eskola Haemophilus influenzae (NTHI):054, 375, 477, 1003, 1008, 1042, 1147, 1231 E. coli DH5α Neidardt, F. C.1996. Roy Curtiss III J. L. Ingraham, E. C. Lin, K. Brooks, B.Magasanik, W. S. Remikoff, M. Riley, S. M. and H. E. Umbarger (ed.), ASMPress. Salmonella typhimurium rfaC; rfa1; rfaP Schnaitman, C. A., and F.D. Klena. 1993. 57: 655-682

TABLE 2 Reactivity of monoclonal antibody B5 with representativeNeisseria meningitidis strains of immunotypes L1-L12 determined by wholecell ELISA, dot blots of lysates, immunofluorescence and confocalmicroscopy. Serogroup: Immuno- Whole cell Immuno- Strain Serotype: typeELISA^(a)(ODA_(405 nm)) Dot Blot^(b) fluorescence^(c) 126E C: 3: P1.5, 2L1 +1.8 +++ + 35E C: 20: )P1.1 L2 −<0.4 − − H44/76 B.15.P1.7, 1 L3 +1.3+++ ++ 89I C: nt: P1.16 L4 −<0.4 − − M98I B: 4: P1.— L5 −<0.4 +/− − M992B: 5: P1.7, 1 L6 −<0.4 +/− − 6155 B: nt: P1.7, 1 L7 +0.8 ++ + M978 B: 8:P1.7, 1 L8 +1.9 +++ ++ 892257 B: 4: P1, 4 L8 +1.9 120M A: 4: P1.10 L9+1.8 +++ + 7880 A: 4: P1: 6 L10 +2.2 +++ + 7889 A: 4: P1.9 L11 +2.0 +++++ 3200 A: 4: P1.9 L12 +2.1 +++ ++ ^(a)Positive reactivity (OD_(A405) >0.4) (+), negative reactivity (OD_(A405) < 0.4) (−) ^(b)Stronglypositive (+++), positive (++), weakly positive (+/−), negative (−).^(c)Strongly positive (++), positive (+), negative (−).

TABLE 3 Correlation between reactivity with monoclonal antibody B5,immunotyping and location of phosphoethanolamine (PEtn) on HepII ofinner core. Position of PEtn on HepII Strain Mab B5 Immuno-type* O-3 O-6MC58 + L3, 7 + − 1000 − NT − − NGE30 − NT − − EG37 − NT − − BZ157^(#) −L2, 5 − + BZ157^(§) + L3, 7 + − NGH38 − L2, 5 − + Abbreviations: NT =non-typable *MN4A8B2 (L3, 7, 9); MN42F12.32 (L2, 5); MN4C1B (L4, 6, 9);MN40G11.7 (L6) ^(#)BZ157 MAb B5 negative variant ^(§)BZ157 MAb B5positive variant

TABLE 4 Negative ion ES-MS data and proposed compositions of0-deacylated LPS from galE capsule-deficient mutant Neisseriameningitidis MAb B5 negative strains. Average mass units were used forcalculation of molecular weight based on proposed composition asfollows: Glc, 162.15; Hep, 192.17; GlcNAc, 203.19; Kdo, 220.18; PEtn,123.05. Observed Ions (m/z) Molecular Mass (Da) Strain (M − 2H)²⁻ (M −H)⁻ Observed Calculated Lipid A^(b) 1000 1213.0 2427.6 2427.7 2427021075 1252.9 2507.8 2507.8 2507.2 1155 1314.5 2630.9 2603.9 2630.3 1278NGH38 1293.8 2589.5 2589.3 2589.3 952 EG327 1151.2 2304.4 2304.4 2304.1952 NGE30 1132.1 — — 2265.1 1075 1396.1 2793.4 2793.7 2792.5 1075 1436.02873.7 2873.9 2872.5 1155 1498.0 2997.2 2997.1 2995.6 1278 BZ157 1274.62551.4 — 2550.3 1075 1314.8 2631.1 2631.2 2630.3 1155 1376.4 2754.42754.5 2753.4 1278 1457.5 2916.6 2916.6 2915.6 1278 Strain ProposedComposition^(a) 1000 2Glc, GlcNAc, 2Hep, 2 Kdo, Lipid A 2Glc, GlcNAc,2Hep, 2 Kdo, Lipid A 2Glc, GlcNAc, 2Hep, 2 Kdo, Lipid A NGH38 3Glc,GlcNAc, 2Hep, PEtn, 2Kd0, Lipid A EG37 2Glc, GlcNAc, 2Hep, 2 Kdo, LipidA NGE30 Glc, GlcNAc, 2Hep. 2Kdo, Lipid A 3Glc, 2GlcNAc, 2Hep, 2 Kdo,Lipid A 3Glc, 2GlcNAc, 2Hep, 2 Kdo, Lipid A 3Glc, 2GlcNAc, 2Hep, 2 Kdo,Lipid A BZ157 2Glc, GlcNAc, 2Hep, PEtn, 2Kd0, Lipid A 2Glc, GlcNAc,2Hep, PEtn, 2Kd0, Lipid A 2Glc, GlcNAc, 2Hep, PEtn, 2Kdo, Lipid A 3Glc,GlcNAc, 2Hep, PEtn, 2Kd0, Lipid A ^(a)Glc, glucose; GlcNAc,N-acetylglucosamine; PEtn, phosphoethanolamine; Hep, heptose; Kdo,3-deoxy-D-manno-octulosonic acid. ^(b)As determined by MS-MS analyses.

Figure Legends Example 1 FIG. 1

Representation of the structure of meningococcal LPS oligosaccharides ofimmunotypes L1-L9. Immunotypes are indicated to the extreme left. Thevertical dotted line marks the junction between the inner corestructures to the right and outer core structures to the left. Theepitope recognized by MAb B5 is indicated in bold (MAb B5 positive).Arabic numerals indicate the linkage between sugars or amino-sugars.Alpha and beta indicate the carbon 1 linkage at the non-reducing end ofthe sugar. Genes for incorporating each of the key sugars oramino-sugars into the LPS oligosaccharide in the biosynthetic pathwayare indicated with arrows indicating where in the pathway the geneproduct is required. Abbreviations include: Kdo,2-keto-2-deoxyoctulosonic acid; PEtn, phosphoethanolamine; Gal,galactose: GLcNAc, N-acetyl glucosamine; Glc, glucose; Hep, Heptose.Immunotype L5 has no PEtn on the second heptose. The gene that adds theglucose to the second heptose (IgtG) is phase variable.

FIG. 2

Cross-reactivity of MAb B5 with selected immunotypes and mutants ofNeisseria meningitidis LPS and O-deacylated (odA) LPS as determined bysolid phase ELISA. LPS glycoforms of immunotypes L2 (35E) (solid blackbars), L3 (H44/76) (open bars), L4 (891) (diagonal line filled bars), L5(M981), L8 (M978) (horizontal line filled bars), wild-type andrespective mutants (galE, IgtA or IgtB), in a native or O-deacylatedform, were coated onto ELISA plates (see methods) and reactivity of MAbB5 determined by standard ELISA (OD_(A450)).

FIG. 3

Space-filling 3-D molecular models of the calculated (MMC) lowest energystates of the core oligosaccharide from galE mutants of (a) L3, (b) L4and (c) L8-dephosphorylated. Kdo moiety indicated in grey is substitutedat the O-5 position by the heptose disaccharide inner core unit (red),HepI provides the point via a glucose residue (dark green) for extensionto give α-chain epitopes, while HepII is substituted by N-acetylglucosamine residue (lighter green) at 0-2. PEtn (brown) is shown in 0-3position in L3 immunotype and 0-6 in L4 immunotype. Colour versions ofthis and the other figures for Example 1 are to be found in Plested etal., 1999 Infect. Immunity 67, 5417-5426.

FIG. 4

Cross-reactivity of MAb B5 with genetically modified L3 LPS andchemically modified L8 LPS from Neisseria meningitidis as determined bysolid phase ELISA. LPS glycoforms of immunotype L8 (M978) (horizontalline filled bars) chemically modified by O-deacylation and HF treatmentand immunotype L3 m44/76) (open bars) galE, icsB, icsA, lis. PB4 mutants(O-deacylated) were coated onto ELISA plates (see methods) andreactivity of MAb B5 determined by standard ELISA (OD_(A410nm)).

FIG. 5.a.

Confocal immunofluorescence microscopy of Neisseria meningitidisorganisms, strain MC58 adherent to human umbilical vein endothelialcells (HUVECs). (a) Fluorescein tagging with rabbit polyclonal antibodyspecific for Group B Neisseria meningitidis capsule. (b) rhodaminetagging of MAb B5, specific for galE LPS (×2400 magnification). Confocalimmunofluorescence microscopy of in vivo grown MC58 organisms stained asdescribed in Plested et al., 1999 Infect. Immunity 67, 5417-5426. (c)anti-capsular antibody (green). (d) MAb B5 (red) (×2400 magnification).

FIG. 6

Silver-stained tricine gels of LPS preparations (10 μg/lane) fromNeisseria meningitidis Group B strains which were not reactive with MAbB5. These LPS preparations were either not treated (−) or treated with(+) neuraminidase to show the presence of sialic acid: a) MAb B5negative strains Lanes 1, 2=NGE30; lanes 3, 4=BZ157; lanes 5, 6=EG328;lanes 7, 8=1000; lanes 9, 10=3906. b) MAb B5 negative strains: Lanes 1,2=EG327; lanes 3.4=NGH38; lanes 5, 6=NGH15; MAb B5 positive strain:lanes 7, 8=MC58. Presence of sialic acid (NeuAc) indicated by +. Thisband was seen in untreated (−) and removed in treated (+) neuraminidasepreparations.

Example 2 Identification of Additional Inner Core Epitopes Introduction

Example 1 identifies an inner core LPS epitope that was accessible andconserved in 70% of a global collection of 104 Neisseria meningitidisstrains representative of all major serogroups (Plested et al., 1999,Infect. Immunity 67, 5417-5426). The epitope recognized by MAb B5 wasidentified in all LPS immunotypes with phosphoethanolamine (PEtn) in the3-position of β-chain heptose (HepII) of inner core LPS. Further workwas carried out to identify additional epitopes, with the aims outlinedin FIG. 4

In summary:

A series of twelve murine monoclonal antibodies (MAbs) were developed atNRC, by using a procedure described previously by us (Plested et al.,1999 Infect. Immunity 67, 5417-5426). except using formalin-fixedNeisseria meningitidis L4 (strain 891) galE whole-cells. The twelve MAbswere extensively screened by ELISA using purified LPS from Neisseriameningitidis mutants and wild-type strains and three MAbs B2 (IgG2b), A4(IgG2a), and A2 IgG2a were chosen for further investigation.Conservation of the inner core LPS epitope was assessed at Oxford usingwild-type whole-cell lysates of a global collection of 104 Neisseriameningitidis disease isolates (Maiden, M. C. J., J. A. Bygraves, E.Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth,D. A. Caugant, L M. Feavers, M. Achtman, and G. B. Spratt. 1998. PNAS95: 3140-3145). Accessibility of the inner core LPS epitope was assessedusing immunofluorescence microscopy with ethanol-fixed Neisseriameningitidis whole-cells of wild type and mutants adherent to amonolayer epithelial cells (Plested et al. 1999).

Each of the three MAbs reacted with purified Neisseria meningitidis L4galE LPS by ELISA. Except for MAb B2 that bad low reactivity withNeisseria meningitidis L4 LPS, none of the Neisseria meningitidis L4series of MAbs were able to the recognize wild-type L4 or L2 purifiedLPS by ELISA. None of the Neisseria meningitidis L4 MAbs recognizedNeisseria meningitidis wild-type L2 or L4 whole-cells byimmunofluorescence microscopy.

MAb B2 reacted with 15/32 Neisseria meningitidis MAb B5 negativeNeisseria meningitidis strains and 9/68 Neisseria meningitidis MAb B5positive Neisseria meningitidis strains by whole-cell dot blot analysis.MAb 2 reacted with L4 galE, L4 wild-type (very low reactivity) but notL3 galE, L2 galE (native) O-deacylated (odA), L2 wild-type (native-odA),L5, L6 wild-type LPS.

MAb A2 recognized 28/32 Neisseria meningitidis MAb B5 negative Neisseriameningitidis strains and 20/68 Neisseria meningitidis MAb B5 positiveNeisseria meningitidis strains by whole-cell dot blot analysis. MAb A2reacted with L4 galE (native/odA), L2 galE (native) but not L3 galE, L2galE (odA), L2 wild-type (native/odA), L4, L5, L6 wild type LPS.

MAb A4 reacted with 29/32 Neisseria meningitidis MAb B5 negativeNeisseria meningitidis strains and 24/68 Neisseria meningitidis MAb B5positive Neisseria meningitidis strains by whole-cell lysate dot blotanalysis. MAb A4 reacted with LA galE, L2 galE (native/odA), button L3galE, L2 wild type, L4, L5, L6, L8 wild-type LPS.

Based on these results, MAb A4 (IgG2a) was chosen for further study asit demonstrated specificity for both L4 galE and L2 galE LPS by ELISAand recognized all except 3 Neisseria meningitidis B5 negative Neisseriameningitidis strains (BZ232 serogroup B; NGH38 serogroup B; F1576serogroup C). Together MAbs B5 and A4 were able to recognize 97/100Neisseria meningitidis isolates. Immunofluorescence microscopydemonstrated that MAb A4 was able to access the inner core epitope in anL4 galE mutant in the presence of capsule.

We have identified LPS inner-core epitopes with PEtn at the 3-positionof HepII (MAb B5) or not at the 3 position (MAb A4). There remain 3strains out of 100 (BZ232, NGH38 and F1576) which show no reactivitywith either MAb A4 or MAb B5. The structural basis for thisnon-reactivity is under investigation. Once all the variant glycoformsof the inner core are known, of which at least 3 have been identified,the rationale will exist for including epitopes, representative of allNeisseria meningitidis strains causing invasive disease, in a conjugatevaccine. This will be tested for proof in principle using studies inanimals before proceeding to human trials.

The following techniques were used:

(1) Murine MAb A4 (IgG2a) was raised to galE (89I, L4 immunotype) andselected on basis of reactivity in LPS ELISA & immunofluorescence (IF)microscopy.

(2) LPS ELISA (Plested et al., 2000 J. Immunol. Meth. 237:73-84):Microtitre plates (Nunc) coated with purified (galE) LPS (10 μg/ml)overnight, were washed, blocked, incubated with MAb for 1 h, washed anddetected with anti-mouse IgG alkaline phosphatase and p-NPP(OD_(A405nm)).

(3) Immunoblotting using whole-cell lysates from 104 Neisseriameningitidis strains (Plested et al., 1999 IAI 67:5417-5426). MAb A4 wasdetected using anti-mouse IgG alkaline phosphatase and BCIP/NBT.

(4) Immunofluorescence microscopy: as before (Plested et al., 1999 IAI67:5417-5426) or Neisseria meningitidis were adherent to cultured humanbuccal epithelial cell line (16HBE140) instead of HUVECs; fixed,blocked, incubated with MAb A4 and anti-capsular serogroup B antibodythen detected using fluorescently labeled secondary antibodies (TRITC orFITC).

(5) Fine structural analysis of purified O-deacylated LPS samples bynegative-ion ES-MS and NMR (Plested et al., 1999 IAI 67:5417-5426).

Results: 1) Accessibility of LPS Epitope in Neisseria MeningitidisWhole-Cells.

See FIG. 7.

MAb A4 accesses the inner core LPS epitope in Neisseria meningitidis L4galE mutant in the presence of capsule (magnification ×100). Neisseriameningitidis L4 galE adherent to epithelial cells (16HBE140) stainedwith:

a. MAb A4 (anti-mouse TRITC-red);b. anti-cap B (anti-rabbit FITC-green);c. triple staining with MAb A4 (anti-mouse TRITC-red), anti-cap B(anti-rabbit FITC-green) and epithelial cells stained DAPI (blue).

MAb B5 accesses inner core LPS epitopes in Neisseria meningitidis L3MC58 (magnification ×2400). Neisseria meningitidis L3 MC58 adherent toHUVECs stained with d. MAb B5 (anti-mouse TRITC-red), e. anti-cap B(anti-rabbit FITC-green) using confocal immunofluorescence microscopy.

2) Conservation of LPS Epitope Across all Serogroup of NeisseriaMeningitidis See FIG. 8.

MAb A4 (diagonal hatched) and MAb B5 (horizontal lines) togetherrecognize all Neisseria meningitidis strains by immunoblotting withwhole-cell lysates, except 3 strains (black arrows) which are underfurther analysis. The dendrogram of genetic relationship of Neisseriameningitidis strains from a global collection was constructed by clusteranalysis following Multi-Locus Sequence Typing (MLST) (Maiden et al.,1998 PNAS 95:3140-3145).

3) Genetically Defined LPS Structure See FIG. 3.

Fine LPS structural details demonstrates conformational effects of PEtnon epitope presented. Space-filling 3-D molecular models of (MonopoliesMonte Carlo) calculated lowest energy states of core LPS from galEmutants a L3; b. L4; c. L8 (dephosphotylated). Kdo in grey, Heptose(Hep) in red, Glucose (Glc) and Glucosamine (GlcNAc) in light and darkergreen, (PEtn) in brown.

Conclusions

Inner core glycoforms have been identified with PEtn in the 3-positionof HepII, an exocyclic position of HepII or absent. This study hasindicated that utilization of MAb A4 in conjunction with MAb B5 enables97% of meningococcal strains to be recognized. These studies thereforeindicate that inner core LPS may have potential as a Neisseriameningitidis serogroup B vaccine.

Example 3 Studies on the Functional Activity of Monoclonal Antibody, MAb.B5, and Inner Core (galE) Lipopolysaccharide Antibodies in Human SerumUsing an Opsonophagocytosis Assay, a Serum Bactericidal Assay and an InVivo Passive Protection Model Introduction

We have generated a monoclonal antibody, MAb B5. This antibody isaccessible to inner core LPS structures in Neisseria meningitidis in thepresence of capsule and is conserved in 70% of a representativecollection of Neisseria meningitidis of all strains and 76% of serogroupB strains (Plested, J. S. et al. (1999) Infect & Immun. 67 (10):5417-5426).

Until now it was not known if antibodies in a natural human infectioncan be specific for MAb B5 epitope and have functional activity.

MAb B5 has been shown to have opsonic and bactericidal activity againstgalE mutant and ability to passively protect infant rats againstchallenge with Neisseria meningitidis galE mutant using an in vivomodel.

Methods

(1) Opsonophagocytosis (OP) assay (Plested et al., 2000b): Briefly,fluorescently labeled ethanol-fixed Neisseria meningitidis MC58 or galEmutant or beads coated with purified galE LPS (10 μg/ml) were opsonisedwith MAb B5 and human complement source diluted in final buffer for 10mins/37° C./500 rpm in microtitre plate. Then human peripheral bloodpolymorphonuclear cells (PMNs) prepared from heparinised donor bloodwere diluted in final buffer and added to each well (1×10⁷ cells/ml) andincubated for a further 10 min/37° C./500 rpm. Reaction mixture wasstopped on ice by addition of 150 μl PBS-EDTA and added to FACS tubecontaining 50 μl Trypan Blue. Mixture was mixed and 10,000 lymphocyteswere analysed on FACScan and Cellquest software. PMNs were analyzed byFSC vs appropriate channels to determine % uptake of fluorescentbacteria by granulocytes and monocytes (% OP activity).

(2) Serum Bactericidal (SB) assay method was adapted from CDC protocolexcept MAb B5 was added to dilutions of human pooled sera and 1000 cfuof Neisseria meningitidis strain and incubation time was 40-45 min at37° C. Briefly, bacteria were grown up onto BHI agar overnight fromfrozen stocks. A suspension of bacteria in PBS-B was measured at OD₂₆₀(1:50 in 1% SDS, 0.1% NaOH). Using a 96-well microtitre plate 50 μlbuffer was added to wells in columns 2-7. 50 μl of 80% decomplementedhuman pooled sera was added to column 8 wells. 100 μl of 80% pooled serawas added to wells in column 1. Two-fold serial dilutions of antibodywere added to columns 1-7 (discarding the last 50 μl from column 7). 50μl of bacterial suspension diluted to give 1000 cfu in 50 μl were addedto wells of columns 1-8. The mixture was incubated for 40-45 minutes andplated out onto BHI agar for overnight incubation. The number ofcolonies on each plate were counted and the results expressed as a % ofcfu/ml in decomplemented control well.

(3) In vivo passive protection model using 5-day old Wistar infant ratsmodel. This model was as described by Moe, G. R., et al., 1999. Infect.Immun. 67: 5664-5675, except higher doses of Neisseria meningitidisbacteria were used and different Neisseria meningitidis strain was used.Briefly, groups of 5 day old infant rats were randomized with mothers.Weighed and given inoculum 1×10⁸ cfu/ml Neisseria meningitidis galEmutant mixed 1:1 with either (i) No antibody (PBS) (ii) Affinitypurified MAb B5 (10 μg) (iii) Affinity purified MAb B5 (100 pg) (iv) MAb735 (anticapsular group B antibody) (2 μg). Infant rats were monitoredfor signs of infection and sampled by tail vein bleed at 6 hourspost-infection. Animals were weighed and terminal bleed was taken after24 h by cardiac puncture following injection of pentobarbitone. Neat anddiluted blood were plated immediately onto BHI plates and incubatedovernight. Plates were counted next day to determine bacteremia (cfu/ml)at 6 h and 24 h.

(4) LPS ELISA (Plested et al., 2000a. Microtitre plates (Nunc) coatedwith purified (galE) LPS (10 μg/ml) overnight, were washed, blocked andincubated with MAb or human sera for 1 h, washed and detected withanti-mouse or anti-human IgG alkaline phosphatase and p-NPP(OD_(A405nm)).

(5) Affinity purified MAb B5. Spent culture supernatant from MAb B5 waspurified on Protein A-sepharose column and eluted with Glycine pH 4.0,neutralized with Tris-HC1 pH9.0. Fractions were tested for reactivity onLPS ELISA, pooled and concentrated using Amicon-filter. Purity wasdetermined by SDS-PAGE gel and protein concentration was determined byOD and protein assay.

6) FACS Surface Labeling of Neisseria meningitidis Bacteria

The method was adapted from Moe et al, (Moe, G. R., et al., 1999. InfectImmun 67: 5664-5675) except no sodium azide was included in the blockingbuffer step (Plested et al., 2000b. To prepare labelled bacteriaNeisseria meningitidis (strain MC58, galE) organisms were grownovernight by standard conditions at 37° C. on BHI agar plates and gentlysuspended in PBS. OD_(A260nm) was adjusted to give the requiredconcentration e.g. 5×10⁹ org./ml. 100 μl bacterial cells were added toeach FACS tube (5×10⁸ org.) and an equal volume of diluted sera (1/100MAb B5 in 1% BSA/PBS) was added. Tubes were incubated for 2 hours at 4°C. and cells centrifuged for 5 minutes at 13,000 g. The supernatant wasdiscarded and cells were washed with 200 μl of 1% BSA/PBS. 100 μl ofFITC-conjugated F(ab)₂ goat anti-mouse (Sigma F2772) was added, diluted1:100 in 1% BSMBS, and tubes were incubated for 1 hour at 4° C. Cellswere centrifuged at 13,000 g for 5 minutes and washed by addition of 200μl of 1% BSMBS. The supernatant was discarded and the cells weresuspended in 1% v/v formaldehyde. Samples were transferred to FACScantubes and analysed on the FACS.

Results 1) Clinical Relevance of MAb B5 Epitope:

We present data on three paired sera taken from infants early (acute)and later (convalescent) during culture confirmed invasive meningococcaldisease (IMD) that resulted from infection with Neisseria meningitidisisolates of immunotypes L1, L3 (MAb B5 reactive) (patients 1 and 2) andL2 immunotype (MAb B5 non-reactive) (patient 3) (FIG. 10). The Neisseriameningitidis isolates for patients 1, 2, 3 were L1 (B nt p1.14), L3 (B15p1.7) and L2 (C2ap1.5) respectively. One paired sera from patient 2infected with a Neisseria meningitidis strain that was MAb B5 reactivedemonstrated an increase in specific inner core LPS antibodies by ELISAbetween early and late infection (p=0.03 not significant two-tailedpaired t-test, 95% CI 0.09-90.8) (FIG. 10 a). Patient 1 serademonstrated no significant difference in the titre of antibody takenearly and later during IMD but the titre of the early sample was alreadyat a high level (FIG. 10 a). The lack of increase may reflect higheraffinity antibody in the convalescent sample that would not be detectedin this ELISA. However in both patient 1 and 2 sera there was a nearlysignificant increase in functional activity in the convalescent sera inan opsonophagocytosis assay with L3 wild-type strain MC58 and humanperipheral polymorphonuclear cells (p=0.06 two-tailed paired t-test, 95%CI 0.90-5.96) (FIG. 10 b) (Plested et al., 2000b). There was nosignificant increase in specific antibody titre between acute andconvalescent sera taken from patient 3 infected with L2 immunotypestrain (MAb B5 non-reactive) as measured by ELISA (FIG. 10 a). There wasno significant functional activity in OP assay against L3 wild-typestrain with sera taken from patient 3 early or later during IMD (FIG. 10b). This demonstrates the clinical relevance of the MAb B5 epitope invivo and that specific inner core LPS antibodies are functional in vivo.

FIG. 10. A. ELISA titres of antibodies to L3 galE LPS (IgG) in pairedsera taken early and late from children with invasive meningococcaldisease.

B. Mean % phagocytosis of Neisseria meningitidis MC58 with paired serataken early and late from children with invasive meningococcal diseasewith human peripheral blood mononuclear cells and human complement.

2) Supporting Evidence that Murine MAb B5 has Functional Activity inBiologically Relevant Assays and an In Vivo Model.

(i) Opsonophagocytosis Assay

The OP assay provides evidence that MAb B5 has opsonic activity againstNeisseria meningitidis wild type and galE mutant and that the OPactivity is specific far MAb B5 epitope.

The specificity of MAb B5 reactivity using wild-type Neisseriameningitidis MC58 was shown by inhibition studies. MAb B5 waspre-incubated with different concentrations of purified LPS. There was adose response inhibition in OP activity with Neisseria meningitidis MC58with increasing concentrations of galE LPS added to MAb B5 (see FIG. 11a).

FIG. 11 a. Mean % phagocytosis of Neisseria meningitidis MC58 with MAbB5 pre-incubated with increasing concentrations of either (i) B5reactive or (ii) B5 non-reactive galE LPS with human peripheral bloodpolymorphonuclear cells and human complement.

MAb B5 has specific OP activity for MAb B5 reactive strains using anisogenic pair of Neisseria meningitidis wild-type strains (Neisseriameningitidis BZ157, serogroup B) that are MAb B5 reactive or MAb B5non-reactive. MAb B5 has opsonic activity with MAb B5 reactive strainbut not MAb B5 non-reactive strain (see FIG. 11 b).

FIG. 11 b. Mean % phagocytosis of pair of Neisseria meningitidiswild-type isogenic strains (Neisseria meningitidis BZ157) that areeither MAb B5 reactive or B5 non-reactive with MAb B5 as the opsoninwith human peripheral blood mononuclear cells and human complement.

OP assay demonstrated the uptake of beads coated with purified L3 galELPS opsonised with MAb B5 was significantly greater than the uptake withuncoated beads. This demonstrates the specificity of MAb B5 for galE LPScoated onto beads (see FIG. 11 c).

FIG. 11 c. Mean % phagocytosis of fluorescent latex beads coated witheither purified LPS from L3 galE mutant (10 μg/ml) or uncoated, in thepresence of MAb B5 or final buffer, with human peripheral bloodmononuclear cells and human complement.

(ii) Serum Bactericidal Assay

The SB assay provides evidence that MAb B5 has bactericidal activityagainst Neisseria meningitidis galE mutant in SB assay in the presenceof a human complement source (see method).

The serum sensitivity of galE mutant with either no antibody or in thepresence of MAb B5 was compared (FIG. 12). There was a dose responseincrease in bactericidal activity of galE mutant shown by decreasing %survival, with decreasing % of serum in the presence of MAb B5 comparedto no antibody.

FIG. 12. Mean % survival of Neisseria meningitidis galE mutant in thepresence and absence of MAb B5 against two-fold serial dilutions ofhuman pooled serum starting at 40% as detected using a serumbactericidal assay (see methods).

(iii) Passive Protection Model Using the Infant Rat.

Using the 5-day-old infant rat model we have demonstrated that two dosesMAb B5 are able to reduce bacteremia against challenge with 1×10⁸ cfu/mlNeisseria meningitidis MC58 galE mutant i.p. compared to no antibodycontrols. This data demonstrates the ability of MAb B5 to passivelyprotect against challenge with Neisseria meningitidis MC58 galE mutantand correlates with the functional activity of MAb B5 in OP and SBassays against the same Neisseria meningitidis strain.

FIG. 13. Geometric mean bacteremia in the blood of groups of 5 day oldinfant rats 24 h post-infection with 1×10⁸ cfu/ml galE mutant givensimultaneously with either: (i) no antibody (ii) MAb B5 (10 μg dose);(iii) MAb B5 (100 μg dose); (iv) MAb 735, a positive controlanti-capsular antibody (2 μg dose).

MAb B5 Binding Studies

Additional evidence that MAb B5 recognises both wild-type and galEmutant LPS is shown in the following binding studies:

a) Western Blot Analysis

Purified LPS from wild type Neisseria meningitidis MC58 and galE mutantwas separated on standard Tricine gel and blotted onto nitrocellulose bystandard methods. The blot was probed with MAb B5 culture ascites(1:2000) overnight and detected using anti-mouse IgG and BCIP/NBTsubstrate. The blot demonstrates binding of MAb B5 to higher molecularweight wild-type LPS band and lower molecular weight galE LPS band inwild-type LPS. This demonstrates that MAb B5 can access and b i d to thewild-type LPS as well as truncated galE LPS.

FIG. 14. Western blot showing purified LPS from Neisseria meningitidisMC58 and galE mutant probed with MAb B5 (ascites fluid 1:2000) detectedusing anti-mouse IgG alkaline phosphatase and BCIP/NBT substrate.

b) FACS Surface Labelling Data

MAb B5 binding to live wild-type strain MC58 and galE mutant (1×10⁸cfu/ml) were quantitatively compared using surface labeling withanti-mouse FITC and analyzed by FACS. The relative binding of MAb B5 toNeisseria meningitidis MC58 was 82.5% and Neisseria meningitidis galEmutant was 96. 9% demonstrating that as expected the greatest bindingwas to the galE mutant but there was still significant binding to thewild-type strain MC58.

FIG. 15. FACS profile comparing surface labeling of live Neisseriameningitidis MC58 and galE mutant (5×10⁸ org./ml) with MAb B5 (culturesupernatant 150) detected using anti-mouse IgG (FITC labeled).

1. A monoclonal antibody binding to an epitope of the lipopolysaccharide(LPS) inner core of a galE mutant strain of Neisseria meningitidis,wherein the LPS inner core epitope comprises a phosphoethanolaminemoiety linked to the HepII of the LPS inner core, and wherein themonoclonal antibody binds to a Neisseria meningitidis strain having animmunotype selected from the group consisting of: L1, L3, L7, L8, L9,L10, L11, and L12, but not having an immunotype selected from the groupconsisting of: L2, L4, L5, and L6, or the monoclonal antibody binds to aNeisseria meningitidis strain having an immunotype selected from thegroup consisting of: L2, L4, L5, and L6, but not having an immunotypeselected from the group consisting of: L1, L3, L7, L8, L9, L10, L11, andL12.
 2. The monoclonal antibody according to claim 1, wherein the LPSinner core epitope comprises a phosphoethanolamine moiety linked to the3 position of HepII of the LPS inner core and the monoclonal antibodybinds to a Neisseria meningitidis strain having an immunotype selectedfrom the group consisting of: L1, L3, L7, L8, L9, L10, L11, and L12, butnot of the lipopolysaccharide inner core of a Neisseria meningitidisstrain having an immunotype selected from the group consisting of: L2,L4, L5, and L6, wherein the monoclonal antibody is monoclonal antibodyB5 produced by the hybridoma deposited with the accession number IDAC260900-1.
 3. The monoclonal antibody according to claim 1, wherein theLPS inner core epitope comprises a phosphoethanolamine moiety linked tothe 6 or 7 position of HepII of the LPS inner core, and wherein themonoclonal antibody binds a Neisseria meningitidis strain having animmunotype selected from the group consisting of: L2, L4, L5, and L6,but not of the lipopolysaccharide inner core of a Neisseria meningitidisstrain having an immunotype selected from the group consisting of: L1,L3, L7, L8, L9, L10, L11, and L12, wherein the monoclonal antibody ismonoclonal antibody A4 produced by the hybridoma deposited with theaccession number IDAC 260900-2.